Crosslinked hyaluronic acid-collagen gels for improving tissue graft viability and soft tissue augmentation

ABSTRACT

Hydrogels comprising a macromolecular matrix and water may be used to augment soft tissue of a human being, promote or support cell or tissue viability or proliferation, create space in tissue, and for other purposes. A macromolecular matrix may comprise a hyaluronic acid component crosslinked to a collagen component.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/686,444, filed on Aug. 25, 2017, which is a continuation of U.S.patent application Ser. No. 15/494,991 filed on Apr. 24, 2017, which isa continuation of U.S. patent application Ser. No. 14/535,033 filed onNov. 6, 2014, which granted as U.S. Pat. No. 9,662,422 on May 30, 2017,which is a continuation of U.S. patent application Ser. No. 13/740,712filed on Jan. 14, 2013 now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 13/728,855, filed Dec. 27, 2012 nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 13/667,581, filed on Nov. 2, 2012 now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 13/605,565,filed on Sep. 6, 2012 now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 13/603,213 filed on Sep. 4, 2012 nowabandoned; U.S. patent application Ser. No. 13/740,712 claims thebenefit of U.S. Provisional Patent Application No. 61/586,589 filed Jan.13, 2012; U.S. patent application Ser. No. 13/728,855 claims the benefitof U.S. Provisional Patent Application No. 61/580,971, filed Dec. 28,2011; U.S. patent application Ser. No. 13/667,581 claims the benefit ofU.S. Provisional Patent Application No. 61/555,970, filed Nov. 4, 2011;U.S. patent application Ser. No. 13/605,565 claims the benefit of U.S.Provisional Patent Application No. 61/531,533, filed Sep. 6, 2011; andU.S. patent application Ser. No. 13/603,213 claims the benefit of U.S.Provisional Patent No. 61/531,533, filed Sep. 6, 2011; the entirecontent of each of these documents is incorporated herein by thisspecific reference.

BACKGROUND

Hyaluronic acid and collagen are key structural components of humantissues. These biopolymers have been widely used to construct tissueengineering scaffolds and materials for cell culturing and regenerativemedicine.

Autologous fat transfer (AFT), also known as fat grafting, is a processby which fat is harvested from one part of a human body and injectedinto another part of the same person's body where additional bulk may beneeded for cosmetic and/or aesthetic purposes. Clinical applications forautologous fat transfer are expanding rapidly with recent reported usein breast reconstruction and augmentation, buttock enhancement,treatment of congenital tissue defects, facial reconstruction, and skinrejuvenation. Although this is a very attractive approach and there isan increased trend in replacement of soft tissue volume with AFT,typical survival rates of grafted fat may be poor and overall resultsmay not be satisfactory to a patient.

The present invention addresses these and other shortcomings in thefield of cosmetic and reconstructive medicine and procedures.

SUMMARY

Hydrogels and hydrogel compositions have been developed that are usefulfor soft tissue augmentation procedures, including tissue reconstructionprocedures. These hydrogels and hydrogel compositions may promote and/orsupport the survival or growth of living cells and other components oftissues.

In one aspect, a soft tissue augmentation product is provided which canbe injected or introduced into tissue along with a cellular component.The product may comprise a forming component comprising a hydrogeldescribed herein, the hydrogel having a form suitable for augmentinghuman soft tissue by introducing, for example, by injection orimplantation, the forming component into the human tissue. In someembodiments, the hydrogel itself contains or includes a cellularmaterial, for example, living tissue or living cells, and a componenthyaluronic acid and collagen. The product may further comprise a labelincluding instructions for such injecting or implanting the formingcomponent. In addition, the product may, in some embodiments, include asyringe or other device for facilitating the introducing of the formingcomponent.

Typically, in accordance with one aspect of the invention, a hydrogel ora hydrogel composition may comprise water, and a crosslinkedmacromolecular matrix. The matrix may be in a form suitable for mixingor combining with living cells or tissue prior to introduction of thematrix into the portion or anatomical feature being augmented. In someembodiments, the matrix comprises a hyaluronic acid component; and acollagen component. In a more specific aspect of the invention, thehyaluronic acid is crosslinked to the collagen, for example, by acrosslinking component. In one especially advantageous embodiment, atleast a portion of the crosslink units of the crosslinking componentcomprises an ester bond or an amide bond.

In another aspect of the invention, methods of augmenting soft tissue ofa human being are provided which comprise injecting or implanting ahydrogel composition described herein into a soft tissue of the humanbeing to thereby augment the soft tissue. In some embodiments, themethod includes combining, or mixing the hydrogel composition withliving cells or tissue that have been explanted from the patient. Thecomposition may be especially effective in enhancing cell proliferationand/or supporting cell viability when reintroduced, for example, into abreast of a patient. Thus, the method in these instances may be usefulin conjunction with fat grafting procedures.

Other aspects of the invention are directed toward methods of promotingor supporting cell proliferation or survival, for example, in fatgrafting procedures or other augmentation or reconstructive procedures.For example, the methods may include contacting hydrogel compositionsdescribed herein with cellular materials, cells and/or tissue, forexample, prior to injecting the compositions into the body.

In yet other aspects of the invention, methods are provides forpreparing a space in human or animal tissue, for example, for laterreceipt of a fat graft or implant, the method comprising injecting ahydrogel composition described herein into the tissue, and allowinggrowth or proliferation of tissue while the composition degrades overtime.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are bright field micrographs of the attachment of cellson a hyaluronic acid-collagen(I) hydrogel of Example 9 (18 hrs) (FIG.1A) as compared to a hyaluronic acid control (FIG. 1B).

FIG. 2. is a bright field micrograph of cell migration from rat aorticrings for HA-collagen hydrogels of Example 10.

FIGS. 3A and 3B are bright field micrographs of the attachment andproliferation of HUVECs on hyaluronic acid-collagen(I) hydrogelaccording to Example 11 (FIG. 3B) as compared to a HA hydrogel control(FIG. 3A).

FIGS. 4A through 4D show photographs as insets and corresponding H&Emicrographs of lipoaspirate/hyaluronic acid-collagen(I) hydrogel graftsaccording to Example 16 (FIGS. 4B, 4C, and 4D) as compared to grafts oflipoaspirate only (FIG. 4A).

DETAILED DESCRIPTION

Hydrogels described herein may be used to augment soft tissue of a humanbeing. For example, a hydrogel or a hydrogel composition may be injectedor implanted a hydrogel composition into a soft tissue of the humanbeing to thereby augment the soft tissue. In some embodiments, a formingcomponent may comprise a hydrogel or a hydrogel having a form suitablefor augmenting human soft tissue by injecting or implanting the formingcomponent into the human tissue.

A forming component may be any object or substance with a form that issuitable for a particular augmentation need. For example, a formingcomponent may have a viscosity, firmness, and/or other physicalproperties such that, when injected or implanted into a soft tissue toaugment the tissue, the newly augmented portion of the tissue isreasonably similar to the natural tissue. If a forming component is tobe injected, it may be in a form that is suitable for injection. Forexample, the viscosity may be low enough so that injection through aneedle is possible. If a forming component is to be implanted, in somecircumstances it may be desirable for the forming component to be solidor sufficiently viscous so as to maintain its shape during implantation.

Some augmentation products may include a label comprising instructionsto inject or implant the forming component into the human tissue.

Hydrogels described herein may also be used to enhance, promote orsupport cell proliferation or survival. Some embodiments include amethod comprising contacting a hydrogel or a hydrogel composition with acell or cells.

A hydrogel or a hydrogel composition that contacts one or more cells maypromote or support survival of the cells, including adipocytes,adipose-derived stem cells, stromal vascular fraction cells, or acombination thereof. For example, a hydrogel or a hydrogel compositiondescribed herein may promote or support cell survival to a greaterextent than a hydrogel composition comprising hyaluronic acid having aweight concentration that is similar to the weight concentration of thecrosslinked macromolecular matrix used in a hydrogel described herein.In some embodiments, a hydrogel or a hydrogel composition describedherein may promote or support cell survival to a greater extent than ahydrogel composition comprising water and hyaluronic acid at aconcentration of about 24 mg/mL or about 16 mg/mL. Contact between ahydrogel or a hydrogel composition described herein and cells maypromote or support cell survival in vivo to a greater extent than ahydrogel composition that is substantially identical except that thehyaluronic acid component and the collagen component are notcrosslinked. In some embodiments, a hydrogel or a hydrogel compositionmay promote or support cell survival about as well as, or better than,tissue culture polystyrene.

A hydrogel composition disclosed herein may enhance survival of one ormore cells. In one embodiment, a hydrogel composition disclosed hereinenhances survival of one or more cells as compared to cells alone. Inaspects of this embodiment, a hydrogel composition disclosed hereinenhances survival of one or more cells by at least about 50% at leastabout 100%, at least about 150%, at least about 200%, at least 250%, atleast about 300%, at least about 350%, at least about 400%, at leastabout 450%, or at least about 500% as compared to cells alone. Inaspects of this embodiment, a hydrogel composition disclosed hereinenhances survival of one or more cells by about 50% to about 250%, about50% to about 500%, about 50% to about 1000%, about 100% to about 300%,about 100% to about 500%, about 150% to about 400%, about 150% to about500%, or about 200% to about 500%, as compared to cells alone.

In some embodiments, a hydrogel composition disclosed herein enhancessurvival of one or more cells as compared to cells with a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the collagen component are not crosslinked. Inaspects of this embodiment, a hydrogel composition disclosed hereinenhances survival of one or more cells by at least about 50% at leastabout 100%, at least about 150%, at least about 200%, at least 250%, atleast about 300%, at least about 350%, at least about 400%, at leastabout 450%, or at least about 500% as compared to cells with a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the collagen component are not crosslinked. Inaspects of this embodiment, a hydrogel composition disclosed hereinenhances survival of one or more cells by about 50% to about 250%, about50% to about 500%, about 50% to about 1000%, about 100% to about 300%,about 100% to about 500%, about 150% to about 400%, about 150% to about500%, or about 200% to about 500% as compared to cells with a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the collagen component are not crosslinked. Inanother aspect of this embodiment, a hydrogel composition that issubstantially identical to a hydrogel composition disclosed hereinexcept that the hyaluronic acid component and the collagen component arenot crosslinked comprises hyaluronic acid at a concentration of about 16mg/mL and water.

In yet another embodiment, a hydrogel composition disclosed hereinenhances survival of one or more cells as compared to cells with ahydrogel composition that is substantially identical except that thecollagen component is absent. In aspects of this embodiment, a hydrogelcomposition disclosed herein enhances survival of one or more cells byat least about 50% at least about 100%, at least about 150%, at leastabout 200%, at least 250%, at least about 300%, at least about 350%, atleast about 400%, at least about 450%, or at least about 500% ascompared to cells with a hydrogel composition that is substantiallyidentical except that the collagen component is absent. In aspects ofthis embodiment, a hydrogel composition disclosed herein enhancessurvival of one or more cells by about 50% to about 250%, about 50% toabout 500%, about 50% to about 1000%, about 100% to about 300%, about100% to about 500%, about 150% to about 400%, about 150% to about 500%,or about 200% to about 500% as compared to cells with a hydrogelcomposition that is substantially identical except that the collagencomponent is absent. In another aspect of this embodiment, a hydrogelcomposition that is substantially identical to a hydrogel compositiondisclosed herein except that the collagen component is absent compriseshyaluronic acid at a concentration of about 16 mg/mL and water.

A hydrogel or a hydrogel composition that contacts one or more cells maypromote or support proliferation of cells, such as regenerative cells,stem cells, progenitor cells, precursor cells, adipose-derived stemcells, stromal vascular fraction cells, etc. A hydrogel or a hydrogelcomposition described herein may also promote or support cellproliferation to a greater extent than a hydrogel composition comprisinghyaluronic acid having a weight concentration that is similar to theweight concentration of the crosslinked macromolecular matrix used in ahydrogel described herein. In some embodiments, a hydrogel or a hydrogelcomposition described herein may promote or support cell proliferationto a greater extent than a hydrogel composition comprising water andhyaluronic acid at a concentration of about 24 mg/mL or about 16 mg/mL.Contact between a hydrogel or a hydrogel composition described hereinand cells may promote or support cell proliferation to a greater extentthan a hydrogel composition that is substantially identical except thatthe hyaluronic acid component and the collagen component are notcrosslinked. In some embodiments, a hydrogel or a hydrogel compositionmay promote or support cell proliferation about as well as, or betterthan, tissue culture polystyrene.

A hydrogel composition disclosed herein may enhance proliferation of oneor more cells. In one embodiment, a hydrogel composition disclosedherein enhances proliferation of one or more cells as compared to cellsalone. In aspects of this embodiment, a hydrogel composition disclosedherein enhances proliferation of one or more cells by at least about 50%at least about 100%, at least about 150%, at least about 200%, at least250%, at least about 300%, at least about 350%, at least about 400%, atleast about 450%, or at least about 500 as compared to cells alone. Inaspects of this embodiment, a hydrogel composition disclosed hereinenhances proliferation of one or more cells by about 50% to about 250%,about 50% to about 500%, about 50% to about 1000%, about 100% to about300%, about 100% to about 500%, about 150% to about 400%, about 150% toabout 500%, or about 200% to about 500%, as compared to cells alone.

In another embodiment, a hydrogel composition disclosed herein enhancesproliferation of one or more cells as compared to cells with a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the collagen component are not crosslinked. Inaspects of this embodiment, a hydrogel composition disclosed hereinenhances proliferation of one or more cells by at least about 50% atleast about 100%, at least about 150%, at least about 200%, at least250%, at least about 300%, at least about 350%, at least about 400%, atleast about 450%, or at least about 500% as compared to cells with ahydrogel composition that is substantially identical except that thehyaluronic acid component and the collagen component are notcrosslinked. In aspects of this embodiment, a hydrogel compositiondisclosed herein enhances proliferation of one or more cells by about50% to about 250%, about 50% to about 500%, about 50% to about 1000%,about 100% to about 300%, about 100% to about 500%, about 150% to about400%, about 150% to about 500%, or about 200% to about 500% as comparedto cells with a hydrogel composition that is substantially identicalexcept that the hyaluronic acid component and the collagen component arenot crosslinked. In another aspect of this embodiment, a hydrogelcomposition that is substantially identical to a hydrogel compositiondisclosed herein except that the hyaluronic acid component and thecollagen component are not crosslinked comprises hyaluronic acid at aconcentration of about 16 mg/mL or about 24 mg/mL and water.

In yet another embodiment, a hydrogel composition disclosed hereinenhances proliferation of one or more cells as compared to cells with ahydrogel composition that is substantially identical except that thecollagen component is absent. In aspects of this embodiment, a hydrogelcomposition disclosed herein enhances proliferation of one or more cellsby at least about 50% at least about 100%, at least about 150%, at leastabout 200%, at least 250%, at least about 300%, at least about 350%, atleast about 400%, at least about 450%, or at least about 500% ascompared to cells with a hydrogel composition that is substantiallyidentical except that the collagen component is absent. In aspects ofthis embodiment, a hydrogel composition disclosed herein enhancesproliferation of one or more cells by about 50% to about 250%, about 50%to about 500%, about 50% to about 1000%, about 100% to about 300%, about100% to about 500%, about 150% to about 400%, about 150% to about 500%,or about 200% to about 500% as compared to cells with a hydrogelcomposition that is substantially identical except that the collagencomponent is absent. In another aspect of this embodiment, a hydrogelcomposition that is substantially identical to a hydrogel compositiondisclosed herein except that the collagen component is absent compriseshyaluronic acid at a concentration of about 16 mg/mL or about 24 mg/mLand water.

A hydrogel or a hydrogel composition of the present disclosure mayinclude a cellular component, for example, components of human adiposetissue, for example, adipose-derived stem cells, stromal vascularfraction cells, etc.

When injected or implanted in vivo, a hydrogel or a hydrogel compositionmay promote cell and/or tissue growth, including growth into the implantmaterial. For example, a hydrogel or hydrogel composition may stimulateangiogenesis, neovascularization, adipogenesis, collagenesis, cellinfiltration, tissue integration, and the like in vivo. In someembodiments, a hydrogel or a hydrogel composition may promote this typeof growth or activity to a greater extent than a hydrogel compositioncomprising hyaluronic acid having a weight concentration that is similarto the weight concentration of the crosslinked macromolecular matrixused in a hydrogel described herein. In some embodiments, a hydrogel ora hydrogel composition may promote this type of growth or activity to agreater extent than a hydrogel composition comprising water andhyaluronic acid at a concentration of about 24 mg/mL or about 16 mg/mL.In some embodiments, a hydrogel or a hydrogel composition may promotethis type of growth or activity to a greater extent than a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the collagen component are not crosslinked.

Once injected or implanted into a soft tissue, a hydrogel compositiondisclosed herein may stimulate angiogenesis, neovascularization,adipogenesis, and/or collagenesis. In an embodiment, a hydrogelcomposition disclosed herein stimulates angiogenesis,neovascularization, adipogenesis, and/or collagenesis to a greaterextent as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the collagencomponent are not crosslinked. In aspects of this embodiment, a hydrogelcomposition disclosed herein angiogenesis, neovascularization,adipogenesis, and/or collagenesis by at least about 50% at least about100%, at least about 150%, at least about 200%, at least 250%, at leastabout 300%, at least about 350%, at least about 400%, at least about450%, at least about 500%, at least about 750%, or at least about 1000%as compared to a hydrogel composition that is substantially identicalexcept that the hyaluronic acid component and the collagen component arenot crosslinked. In aspects of this embodiment, a hydrogel compositiondisclosed herein angiogenesis, neovascularization, adipogenesis, and/orcollagenesis by about 50% to about 250%, about 50% to about 500%, about50% to about 1000%, about 100% to about 300%, about 100% to about 500%,about 100% to about 1000%, about 150% to about 400%, about 150% to about600%, about 150% to about 1000%, about 200% to about 500%, about 200% toabout 700%, or about 200% to about 1000% as compared to a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the collagen component are not crosslinked. Inanother aspect of this embodiment, a hydrogel composition that issubstantially identical to a hydrogel composition disclosed hereinexcept that the hyaluronic acid component and the collagen component arenot crosslinked comprises hyaluronic acid at a concentration of about 16mg/mL or about 24 mg/mL and water.

In another embodiment, a hydrogel composition disclosed hereinstimulates angiogenesis, neovascularization, adipogenesis, and/orcollagenesis to a greater extent as compared to adipose tissue with ahydrogel composition that is substantially identical except that thecollagen component is absent. In aspects of this embodiment, a hydrogelcomposition disclosed herein angiogenesis, neovascularization,adipogenesis, and/or collagenesis by at least about 50% at least about100%, at least about 150%, at least about 200%, at least 250%, at leastabout 300%, at least about 350%, at least about 400%, at least about450%, at least about 500%, at least about 750%, or at least about 1000%as compared to a hydrogel composition that is substantially identicalexcept that the collagen component is absent. In aspects of thisembodiment, a hydrogel composition disclosed herein angiogenesis,neovascularization, adipogenesis, and/or collagenesis by about 50% toabout 250%, about 50% to about 500%, about 50% to about 1000%, about100% to about 300%, about 100% to about 500%, about 100% to about 1000%,about 150% to about 400%, about 150% to about 600%, about 150% to about1000%, about 200% to about 500%, about 200% to about 700%, or about 200%to about 1000% as compared to a hydrogel composition that issubstantially identical except that the collagen component is absent. Inanother aspect of this embodiment, a hydrogel composition that issubstantially identical to a hydrogel composition disclosed hereinexcept that the collagen component is absent comprises hyaluronic acidat a concentration of about 16 mg/mL or about 24 mg/mL and water.

Once injected or implanted into a soft tissue, a hydrogel compositiondisclosed herein may show infiltration and/or tissue integration ofcells from the soft tissue. In an embodiment, a hydrogel compositiondisclosed herein shows cell infiltration and/or tissue integration fromthe soft tissue to a greater extent as compared to a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the collagen component are not crosslinked. Inaspects of this embodiment, a hydrogel composition disclosed hereinshows enhanced cell infiltration and/or tissue integration by at leastabout 5% at least about 10%, at least about 15%, at least about 20%, atleast 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, or at least about 50% as compared to a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the collagen component are not crosslinked. Inaspects of this embodiment, a hydrogel composition disclosed hereinshows enhanced cell infiltration and/or tissue integration by about 5%to about 25%, about 5% to about 50%, about 10% to about 30%, about 10%to about 50%, about 15% to about 40%, about 15% to about 50%, or about20% to about 50% as compared to a hydrogel composition that issubstantially identical except that the hyaluronic acid component andthe collagen component are not crosslinked. In another aspect of thisembodiment, a hydrogel composition that is substantially identical to ahydrogel composition disclosed herein except that the hyaluronic acidcomponent and the collagen component are not crosslinked compriseshyaluronic acid at a concentration of about 16 mg/mL or about 24 mg/mLand water.

In another embodiment, a hydrogel composition disclosed herein may showcell infiltration and/or tissue integration from the soft tissue to agreater extent as compared to a hydrogel composition that issubstantially identical except that the collagen component is absent. Inaspects of this embodiment, a hydrogel composition disclosed hereinshows enhanced cell infiltration and/or tissue integration by at leastabout 5% at least about 10%, at least about 15%, at least about 20%, atleast 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, or at least about 50% as compared to a hydrogelcomposition that is substantially identical except that the collagencomponent is absent. In aspects of this embodiment, a hydrogelcomposition disclosed herein shows enhanced cell infiltration and/ortissue integration by about 5% to about 25%, about 5% to about 50%,about 10% to about 30%, about 10% to about 50%, about 15% to about 40%,about 15% to about 50%, or about 20% to about 50% as compared to ahydrogel composition that is substantially identical except that thecollagen component is absent. In another aspect of this embodiment, ahydrogel composition that is substantially identical to a hydrogelcomposition disclosed herein except that the collagen component isabsent comprises hyaluronic acid at a concentration of about 16 mg/mL orabout 24 mg/mL and water.

In some methods, hydrogel or a hydrogel composition may be mixed withtissue, for example, adipose tissue or fat tissue from the human being,such as human lipoaspirate, or from fat from another human being or ananimal. The ratio of hydrogel to fat in such a mixture may vary toprovide the desired results. The fat:hydrogel ratio is the weight of thefat divided by the weight of hydrogel. For example, if 1 gram of fat ismixed with 10 grams of hydrogel, the fat:hydrogel weight ratio is 0.1.In some embodiments, the fat tissue and the hydrogel may have afat:hydrogel weight ratio of about 0.1 up to about 10. All otherfat:hydrogel weight ratios falling within this range are alsocontemplated and considered to be within the scope of the presentinvention. For example, the weight ratio may be about 0.5 up to about 7,for example, about 1 up to about 5. In some embodiments, thefat:hydrogel weight ratio is about 1 to about 3, for example, about 1,about 2, or about 3.

A combination or mixture of human fat tissue and hydrogel compositionmay then be injected or implanted into soft tissue of a human being, foraugmenting the breast for example. This may help to improve the survivaltime of grafted fat in autologous and other fat transfer procedures. Itmay also help to improve volume retention, reduce the variability inretained fat graft volume, and/or reduce inflammation as compared toinjecting fat tissue alone.

A hydrogel composition disclosed herein may show improved volumeretention after injection or implantation into a soft tissue. In anembodiment, a hydrogel composition disclosed herein shows improvedvolume retention after injection or implantation into a soft tissue ascompared to a hydrogel composition that is substantially identicalexcept that the hyaluronic acid component and the collagen component arenot crosslinked. In aspects of this embodiment, a hydrogel compositiondisclosed herein shows improved volume retention after injection orimplantation into a soft tissue by at least about 5% at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,or at least about 50% as compared to a hydrogel composition that issubstantially identical except that the hyaluronic acid component andthe collagen component are not crosslinked. In aspects of thisembodiment, a hydrogel composition disclosed herein shows improvedvolume retention after injection or implantation into a soft tissue byabout 5% to about 25%, about 5% to about 50%, about 10% to about 30%,about 10% to about 50%, about 15% to about 40%, about 15% to about 50%,or about 20% to about 50% as compared to a hydrogel composition that issubstantially identical except that the hyaluronic acid component andthe collagen component are not crosslinked. In another aspect of thisembodiment, a hydrogel composition that is substantially identical to ahydrogel composition disclosed herein except that the hyaluronic acidcomponent and the collagen component are not crosslinked compriseshyaluronic acid at a concentration of about 16 mg/mL or about 24 mg/mLand water.

In another embodiment, a hydrogel composition disclosed herein showsimproved volume retention after injection or implantation into a softtissue as compared to a hydrogel composition that is substantiallyidentical except that the collagen component is absent. In aspects ofthis embodiment, a hydrogel composition disclosed herein shows improvedvolume retention after injection or implantation into a soft tissue byat least about 5% at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, or at least about 50% as comparedto a hydrogel composition that is substantially identical except thatthe collagen component is absent. In aspects of this embodiment, ahydrogel composition disclosed herein shows improved volume retentionafter injection or implantation into a soft tissue by about 5% to about25%, about 5% to about 50%, about 10% to about 30%, about 10% to about50%, about 15% to about 40%, about 15% to about 50%, or about 20% toabout 50% as compared to a hydrogel composition that is substantiallyidentical except that the collagen component is absent. In anotheraspect of this embodiment, a hydrogel composition that is substantiallyidentical to a hydrogel composition disclosed herein except that thecollagen component is absent comprises hyaluronic acid at aconcentration of about 16 mg/mL or about 24 mg/mL and water.

A hydrogel composition disclosed herein may show decreased variabilityin volume retention after injection or implantation into a soft tissue.In an embodiment, a hydrogel composition disclosed herein showsdecreased variability in volume retention after injection orimplantation into a soft tissue as compared to a hydrogel compositionthat is substantially identical except that the hyaluronic acidcomponent and the collagen component are not crosslinked. In aspects ofthis embodiment, a hydrogel composition disclosed herein shows decreasedvariability in volume retention after injection or implantation into asoft tissue by at least about 5% at least about 10%, at least about 15%,at least about 20%, at least 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, or at least about 50% ascompared to a hydrogel composition that is substantially identicalexcept that the hyaluronic acid component and the collagen component arenot crosslinked. In aspects of this embodiment, a hydrogel compositiondisclosed herein shows decreased variability in volume retention afterinjection or implantation into a soft tissue by about 5% to about 25%,about 5% to about 50%, about 10% to about 30%, about 10% to about 50%,about 15% to about 40%, about 15% to about 50%, or about 20% to about50% as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the collagencomponent are not crosslinked. In another aspect of this embodiment, ahydrogel composition that is substantially identical to a hydrogelcomposition disclosed herein except that the hyaluronic acid componentand the collagen component are not crosslinked comprises hyaluronic acidat a concentration of about 16 mg/mL or about 24 mg/mL and water.

In another embodiment, a hydrogel composition disclosed herein showsdecreased variability in volume retention after injection orimplantation into a soft tissue as compared to a hydrogel compositionthat is substantially identical except that the collagen component isabsent. In aspects of this embodiment, a hydrogel composition disclosedherein shows decreased variability in volume retention after injectionor implantation into a soft tissue by at least about 5% at least about10%, at least about 15%, at least about 20%, at least 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,or at least about 50% as compared to a hydrogel composition that issubstantially identical except that the collagen component is absent. Inaspects of this embodiment, a hydrogel composition disclosed hereinshows decreased variability in volume retention after injection orimplantation into a soft tissue by about 5% to about 25%, about 5% toabout 50%, about 10% to about 30%, about 10% to about 50%, about 15% toabout 40%, about 15% to about 50%, or about 20% to about 50% as comparedto a hydrogel composition that is substantially identical except thatthe collagen component is absent. In another aspect of this embodiment,a hydrogel composition that is substantially identical to a hydrogelcomposition disclosed herein except that the collagen component isabsent comprises hyaluronic acid at a concentration of about 16 mg/mL orabout 24 mg/mL and water.

Once injected or implanted into a soft tissue, a hydrogel compositiondisclosed herein may reduce inflammation of the soft tissue. In anembodiment, a hydrogel composition disclosed herein reduces inflammationof the soft tissue as compared to a hydrogel composition that issubstantially identical except that the hyaluronic acid component andthe collagen component are not crosslinked. In aspects of thisembodiment, a hydrogel composition disclosed herein reduces inflammationof the soft tissue by at least about 5% at least about 10%, at leastabout 15%, at least about 20%, at least 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, or at leastabout 50% as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the collagencomponent are not crosslinked. In aspects of this embodiment, a hydrogelcomposition disclosed herein reduces inflammation of the soft tissue byabout 5% to about 25%, about 5% to about 50%, about 10% to about 30%,about 10% to about 50%, about 15% to about 40%, about 15% to about 50%,or about 20% to about 50% as compared to a hydrogel composition that issubstantially identical except that the hyaluronic acid component andthe collagen component are not crosslinked. In another aspect of thisembodiment, a hydrogel composition that is substantially identical to ahydrogel composition disclosed herein except that the hyaluronic acidcomponent and the collagen component are not crosslinked compriseshyaluronic acid at a concentration of about 16 mg/mL or about 24 mg/mLand water.

In another embodiment, a hydrogel composition disclosed herein reducesinflammation of the soft tissue as compared to a hydrogel compositionthat is substantially identical except that the collagen component isabsent. In aspects of this embodiment, a hydrogel composition disclosedherein reduces inflammation of the soft tissue by at least about 5% atleast about 10%, at least about 15%, at least about 20%, at least 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, or at least about 50% as compared to a hydrogel compositionthat is substantially identical except that the collagen component isabsent. In aspects of this embodiment, a hydrogel composition disclosedherein reduces inflammation of the soft tissue by about 5% to about 25%,about 5% to about 50%, about 10% to about 30%, about 10% to about 50%,about 15% to about 40%, about 15% to about 50%, or about 20% to about50% as compared to a hydrogel composition that is substantiallyidentical except that the collagen component is absent. In anotheraspect of this embodiment, a hydrogel composition that is substantiallyidentical to a hydrogel composition disclosed herein except that thecollagen component is absent comprises hyaluronic acid at aconcentration of about 16 mg/mL or about 24 mg/mL and water.

A hydrogel or a hydrogel composition may have improved physicalproperties that may help to encourage cell survival or proliferation. Insome embodiments, a hydrogel or a hydrogel composition may allowdiffusion of adipose tissue-specific growth factors or pro-angiogenicgrowth factors to a greater extent than a hydrogel compositioncomprising hyaluronic acid at a concentration of about 24 mg/mL or about16 mg/mL and water.

A hydrogel composition disclosed herein may show improved diffusion ofadipose tissue-specific growth factors or pro-angiogenic growth factors.In an embodiment, a hydrogel composition disclosed herein showsdiffusion of adipose tissue-specific growth factors or pro-angiogenicgrowth factors to a greater extent as compared to a hydrogel compositionthat is substantially identical except that the hyaluronic acidcomponent and the collagen component are not crosslinked. In aspects ofthis embodiment, a hydrogel composition disclosed herein shows improveddiffusion of adipose tissue-specific growth factors or pro-angiogenicgrowth factors by at least about 25% at least about 50%, at least about75%, at least about 100%, at least 125%, at least about 150%, at leastabout 175%, at least about 200%, at least about 225%, or at least about250% as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the collagencomponent are not crosslinked. In aspects of this embodiment, a hydrogelcomposition disclosed herein shows improved diffusion of adiposetissue-specific growth factors or pro-angiogenic growth factors by about25% to about 100%, about 25% to about 150%, about 25% to about 250%,about 50% to about 100%, about 50% to about 150%, or about 50% to about250% as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the collagencomponent are not crosslinked. In another aspect of this embodiment, ahydrogel composition that is substantially identical to a hydrogelcomposition disclosed herein except that the hyaluronic acid componentand the collagen component are not crosslinked comprises hyaluronic acidat a concentration of about 16 mg/mL or about 24 mg/mL and water.

In another embodiment, a hydrogel composition disclosed herein showsdiffusion of adipose tissue-specific growth factors or pro-angiogenicgrowth factors to a greater extent as compared to a hydrogel compositionthat is substantially identical except that the collagen component isabsent. In aspects of this embodiment, a hydrogel composition disclosedherein shows improved diffusion of adipose tissue-specific growthfactors or pro-angiogenic growth factors by at least about 25% at leastabout 50%, at least about 75%, at least about 100%, at least 125%, atleast about 150%, at least about 175%, at least about 200%, at leastabout 225%, or at least about 250% as compared to a hydrogel compositionthat is substantially identical except that the collagen component isabsent. In aspects of this embodiment, a hydrogel composition disclosedherein shows improved diffusion of adipose tissue-specific growthfactors or pro-angiogenic growth factors by about 25% to about 100%,about 25% to about 150%, about 25% to about 250%, about 50% to about100%, about 50% to about 150%, or about 50% to about 250% as compared toa hydrogel composition that is substantially identical except that thecollagen component is absent. In another aspect of this embodiment, ahydrogel composition that is substantially identical to a hydrogelcomposition disclosed herein except that the collagen component isabsent comprises hyaluronic acid at a concentration of about 16 mg/mL orabout 24 mg/mL and water.

A hydrogel or a hydrogel composition may be used to prepare a space inhuman or animal tissue. This may be done by injecting a hydrogel or ahydrogel composition into the tissue. After being injected, a hydrogelor a hydrogel composition may degrade over time, such as over a periodof about 1 week to about 3 months or about 2 weeks to about 6 weeks, tothereby create the space in the tissue. This may create a fertilenutrient bed through stimulated angiogenesis, cellular ingrowth,secretion of tropic factors, as well as creating space. An anestheticmay also be injected into the tissue, such as before injection of ahydrogel or hydrogel composition, or as part of a hydrogel composition.This may help reduce the pain of injection and allow the procedure to bedone as an outpatient procedure.

Once a hydrogel has degraded sufficiently to create a desired space, ahuman or animal fat composition may be injected into the space in thetissue. A fertile nutrient bed created as described above may help toimprove overall fat graft retention as compared to injecting fat withoutpreparing a space as described above.

Some embodiments include a packaged product comprising a device forfacilitating introduction, for example, a syringe loaded with a hydrogeland a needle. A syringe may be fitted with a needle of any size that isappropriate for injecting the hydrogel into the soft tissue of interest,such as a needle with about a #25, about a #30, or a larger gauge.

A filler comprising a hydrogel may be suitable for injection if it canbe injected into the soft tissue of interest without unreasonabledifficulty, and includes fillers that can be dispensed from syringeshaving gauge as low as about #30 or about #25 under normal manualpressure with a smooth extrusion plateau.

Injection of a hydrogel may provide a soft tissue augmentation thatmimics the natural components of the skin. A hydrogel may be injectedintradermally or subcutaneously to augment soft tissue and to repair orcorrect congenital anomalies, acquired defects, or cosmetic defects.Examples of such conditions include congenital anomalies such ashemifacial microsomia, malar and zygomatic hypoplasia, unilateralmammary hypoplasia, pectus excavatum, pectoralis agenesis (Poland'sanomaly), and velopharyngeal incompetence secondary to cleft palaterepair or submucous cleft palate (as a retropharyngeal implant);acquired defects (post traumatic, post surgical, or post infectious)such as depressed scars, subcutaneous atrophy (e.g., secondary todiscoid lupus erythematosus), keratotic lesions, enophthalmos in theunucleated eye (also superior sulcus syndrome), acne pitting of theface, linear scleroderma with subcutaneous atrophy, saddle-nosedeformity, Romberg's disease, and unilateral vocal cord paralysis; andcosmetic defects such as glabellar frown lines, deep nasolabial creases,circum-oral geographical wrinkles, sunken cheeks, and mammaryhypoplasia.

Hydrogels of crosslinked hyaluronic acid alone, collagen, andcrosslinked collagen alone, such as those used in dermal fillers, do notactively promote cellular infiltration and tissue in-growth. Similarly,collagen simply blended into hyaluronic acid hydrogels does not promotetissue integration or de novo tissue generation. However, some hydrogelsdescribed herein do promote cellular migration into the hydrogels andtissue formation within the gels when implanted in vivo.

A hydrogel may comprise water and a crosslinked macromolecular matrix.Typically, a crosslinked molecular matrix may comprise a hyaluronic acidcomponent and a collagen component, wherein the hyaluronic acidcomponent is crosslinked to the collagen component by a crosslinkingcomponent. A crosslinking component may comprise a plurality ofcrosslink units, wherein at least a portion of the crosslink unitscomprise an ester bond or an amide bond.

A hydrogel or a hydrogel composition may be at least about 70%, about93%, or about 96% water by weight, and may approach 100% water byweight. A crosslinked macromolecular matrix may be about 0.01% to about30%, about 0.1% to about 7%, or about 0.2% to about 4% of the weight ofa hydrogel or a hydrogel composition. A hyaluronic acid component may beabout 0.005% to about 20%, about 0.1% to about 5% or about 0.2% to about2.5% of the total weight of a hydrogel or a hydrogel composition. Acollagen component may be about 0.01% to about 10%, about 0.03% to about2%, or about 0.05% to about 1.2% of the total weight of a hydrogel or ahydrogel composition.

A crosslinked macromolecular matrix for a hydrogel may be synthesized bycoupling a hyaluronic acid with a collagen using a coupling agent, suchas a carbodiimide. In these hydrogels, hyaluronic acid may serve as abiocompatible water-binding component, providing bulk and isovolumetricdegradation. Additionally, collagen may impart cell adhesion andsignaling domains to promote cell attachment, migration, and other cellfunctions such as extra-cellular matrix deposition. The biopolymers formhomogeneous hydrogels with tunable composition, swelling, and mechanicalproperties. Compositions can be made to be injectable for minimallyinvasive implantation through syringe and needle.

Hyaluronic acid is a non-sulfated glycosaminoglycan that enhances waterretention and resists hydrostatic stresses. It is non-immunogenic andcan be chemically modified in numerous fashions. Hyaluronic acid may beanionic at pH ranges around or above the pKa of its carboxylic acidgroups. Unless clearly indicated otherwise, reference to hyaluronic acidherein may include its fully protonated, or nonionic form as depictedbelow, as well as any anionic forms and salts of hyaluronic acid, suchas sodium salts, potassium salts, lithium salts, magnesium salts,calcium salts, etc.

Collagen is a protein that forms fibrils and sheets that bear tensileloads. Collagen also has specific integrin-binding sites for celladhesion and is known to promote cell attachment, migration, andproliferation. Collagen may be positively charged because of its highcontent of basic amino acid residues such as arginine, lysine, andhydroxylysine. Unless clearly indicated otherwise, reference to collagenherein may include uncharged collagen, as well as any cationic forms,anionic forms, or salts of collagen.

Because hyaluronic acid may be anionic and collagen may be cationic, thetwo macromolecules may form polyionic complexes in aqueous solution. Apolyionic complex may be significantly less soluble in water than eitherhyaluronic acid or collagen, and thus may precipitate out of aqueoussolution when the two macromolecules are together in a mixture.Furthermore, collagens are often soluble only at low pH and mayprecipitate from solution when brought to a pH amenable to carbodiimidecoupling.

Under certain conditions, a hyaluronic acid and a collagen may becombined in an aqueous liquid in which both components are soluble. Ahyaluronic acid and a collagen may then be crosslinked while both aredissolved in an aqueous solution to form a hydrogel. Reaction conditionssuch as the concentration of hyaluronic acid, the concentration ofcollagen, the pH of the solution, and salt concentration may be adjustedto help to prevent polyionic complex formation between anionichyaluronic acid and cationic collagen. They may also help to preventcollagen microfibril formation, which results in precipitation fromsolution and may prevent crosslinking.

Some embodiments include a method of crosslinking hyaluronic acid andcollagen. This method generally comprises a dissolution step whichresults in an aqueous pre-reaction solution. In a dissolution step,hyaluronic acid and collagen are dissolved in an aqueous solution thathas a low pH and/or a salt to form an aqueous pre-reaction solution.

A hyaluronic acid-collagen crosslinking method further comprises anactivation step. In an activation step, an aqueous pre-reaction solutionis modified by at least adding a water soluble coupling agent and/or byincreasing the pH of the solution. If needed, a salt may also be addedto keep the hyaluronic acid and collagen in solution at the higher pH.Thus, a crosslinking reaction mixture comprises hyaluronic acid andcollagen dissolved or dispersed in an aqueous medium, a water solublecoupling agent, and a salt, and has a higher pH than the aqueouspre-reaction solution from which it was derived. The crosslinkingreaction mixture is allowed to react to thereby crosslink the hyaluronicacid and the collagen.

In some embodiments, the pH of the aqueous pre-reaction solution may beincreased and a substantial amount of fiber formation may be allowed tooccur in the solution before adding the water soluble coupling agent. Insome embodiments, the water soluble coupling agent may be added to theaqueous pre-reaction solution before substantially any fiber formationoccurs.

A crosslinking reaction mixture can react to form a crosslinkedmacromolecular matrix. Since reaction occurs in an aqueous solution, acrosslinked macromolecular matrix may be dispersed in an aqueous liquidin hydrogel form as it is formed by a crosslinking reaction. Acrosslinked macromolecular matrix may be kept in hydrogel form because,in many instances, a crosslinked macromolecular matrix may be used inhydrogel form.

In some embodiments, an aqueous pre-reaction solution or a crosslinkingreaction mixture may further comprise about 10% to about 90% of anorganic solvent in which hyaluronic acid has poor solubility, such asethanol, methanol, isopropanol, or the like.

After a crosslinking reaction has occurred, the crosslinkedmacromolecular matrix may be particulated or homogenized through a mesh.This may help to form an injectable slurry or hydrogel. A mesh used forparticulating a crosslinked macromolecular matrix may have any suitablepore size depending upon the size of particles desired. In someembodiments, the mesh may have a pore size of about 10 microns to about100 microns, about 50 microns to about 70 microns, or about 60 microns.

A hydrogel comprising a crosslinked molecular matrix may be treated bydialysis for sterilization or other purposes. Dialysis may be carriedout by placing a semipermeable membrane between the hydrogel and anotherliquid so as to allow the hydrogel and the liquid to exchange moleculesor salts that can pass through the membrane.

A dialysis membrane may have a molecular weight cutoff that may vary.For example, the cutoff may be about 5,000 daltons to about 100,000daltons, about 10,000 daltons to about 30,000 daltons, or about 20,000daltons.

The dialysis may be carried out against a buffer solution, meaning thatthe liquid on the other side of the membrane from the hydrogel may be abuffer solution. In some embodiments, the buffer solution may be asterile phosphate buffer solution that may comprise phosphate buffer,potassium chloride, and/or sodium chloride. A sterile phosphate buffersolution may be substantially isosmotic with respect to humanphysiological fluid. Thus, when dialysis is complete, the liquidcomponent of a hydrogel may be substantially isosmotic with respect tohuman physiological fluid.

In some embodiments, a crosslinked macromolecular complex may furthercomprise an aqueous liquid. For example, the crosslinked macromolecularcomplex may absorb the aqueous liquid so that a hydrogel is formed. Anaqueous liquid may comprise water with a salt dissolved in it, such as aphosphate buffer, sodium chloride, potassium chloride, etc. In someembodiments, an aqueous liquid may comprise water, sodium chloride at aconcentration of about 100 mM to about 200 mM, potassium chloride at aconcentration of about 2 mM to about 3 mM, and phosphate buffer at aconcentration of about 5 mM to about 15 mM, wherein the pH of the liquidis about 7 to about 8.

In some embodiments, an anesthetic may be included in any compositioncomprising a crosslinked macromolecular complex in an amount effectiveto mitigate pain experienced upon injection of the composition. Examplesof an anesthetic may include, but are not limited to, ambucaine,amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphetamine,bupivacaine, butacaine, butamben, butanilicaine, butethamine,butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine,cyclomethycaine, dibucaine, dimethisoquin, dimethocaine, diperodon,dicyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine,beta-eucaine, euprocin, fenalcomine, fomocaine, hexylcaine,hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate,levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methylchloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine,parethoxycaine, phenacaine, phenol, piperocaine, piridocaine,polidocanol, pramoxine, prilocaine, procaine, propanocaine,proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine,ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine,zolamine, and salts thereof. In some embodiments, the at least oneanesthetic agent is lidocaine, such as in the form of lidocaine HCl. Theconcentration of lidocaine may vary. For example, some compositions mayhave about 0.1% to about 5%, about 0.2% to about 1.0%, or about 0.3%lidocaine by weight (w/w %) of the composition. The concentration oflidocaine in the compositions described herein can be therapeuticallyeffective meaning the concentration may be adequate to provide atherapeutic benefit without inflicting harm to the patient.

A hydrogel may be used in a soft tissue aesthetic product. An aestheticproduct includes any product that improves any aesthetic property of anypart of an animal or human being. A soft tissue aesthetic product maycomprise: an aesthetic device having a form suitable for injecting orimplanting into human tissue; and a label comprising instructions toinject or implant the aesthetic component into human tissue; wherein theaesthetic device comprises a crosslinked macromolecular matrix describedherein. Some products may comprise the crosslinked macromolecular matrixin hydrogel form.

Some embodiments include a method of improving an aesthetic quality ofan anatomic feature of a human being. Improving an aesthetic quality ofan anatomic feature of a human being includes improving any kind ofaesthetic quality including appearance, tactile sensation, etc., andimproving any anatomical feature, including those of the face, limbs,breasts, buttocks, hands, etc. Such a method may comprise injecting orimplanting an aesthetic device into a tissue of the human being tothereby improve the aesthetic quality of the anatomic feature; whereinthe aesthetic device comprises a crosslinked macromolecular matrixcomposition described herein. In some embodiments, the crosslinkedmacromolecular matrix used in the product may be in hydrogel form.

In some embodiments, a hydrogel of a crosslinked macromolecular complexmay have a storage modulus of about 1 Pa to about 10,000 Pa, about 50 Pato 10,000 Pa, about 50 Pa to about 6000 Pa, about 80 Pa to about 2000Pa, about 500 Pa to about 1000 Pa, about 500 Pa to about 4000 Pa, about500 Pa to about 5000 Pa, about 556 Pa, about 560 Pa, about 850 Pa, about852 Pa, about 1000 Pa, or any value in a range bounded by, or between,any of these values.

In some embodiments, a hydrogel of a crosslinked macromolecular complexmay have a loss modulus of about 1 Pa to about 500 Pa, about 10 Pa to200 Pa, about 100 Pa to about 200 Pa, about 20 Pa, about 131 Pa, about152 Pa, or any value in a range bounded by, or between, any of thesevalues.

In some embodiments, a hydrogel of a crosslinked macromolecular complexmay have an average extrusion force of about 10 N to about 50 N, about20 N to 30 N, or about 25 N, when the hydrogel is forced through a 30 Gneedle syringe by moving the plunger of a 1 mL syringe containing thehydrogel at a rate of 100 mm/min for about 11 mm, and measuring theaverage force from about 4 mm to about 10 mm.

A crosslinked macromolecular matrix may have tunable swelling propertiesbased on reaction conditions and hydrogel dilution. In some embodiments,a crosslinked macromolecular matrix may have a swelling ratio of about20 to about 200. A swelling ratio is the ratio of the weight of thecrosslinked macromolecular matrix after synthesis to the weight of thecrosslinked macromolecular matrix without any water. The crosslinkedmacromolecular matrix may have a swelling power of about 1 to about 7.The swelling power is the ratio of the weight of the crosslinkedmacromolecular matrix when it is saturated with water to the weight ofthe crosslinked macromolecular matrix after synthesis.

In a crosslinking reaction, the molecular weight of a hyaluronic acidmay vary. In some embodiments, a hyaluronic acid may have a molecularweight of about 200,000 daltons to about 10,000,000 daltons, about500,000 daltons to about 10,000,000 daltons, about 1,000,000 daltons toabout 5,000,000 daltons, or about 1,000,000 daltons to about 3,000,000daltons. When the crosslinking reaction occurs, the resultingcrosslinked macromolecular product may have a hyaluronic acid componentderived from the hyaluronic acid in the crosslinking reaction. Thus, theranges recited above may also apply to the molecular weight of ahyaluronic acid component, e.g. about 200,000 daltons to about10,000,000 daltons, about 500,000 daltons to about 10,000,000 daltons,about 1,000,000 daltons to about 5,000,000 daltons, or about 1,000,000daltons to about 3,000,000 daltons. The term “molecular weight” isapplied in this situation to a portion of the matrix even though thehyaluronic acid component may not actually be a separate molecule due tothe crosslinking. In some embodiments, a higher molecular weighthyaluronic acid may result in a crosslinked molecular matrix that mayhave a higher bulk modulus and/or less swelling.

The concentration of hyaluronic acid in an aqueous pre-reaction solutionor a crosslinking reaction mixture may vary. In some embodiments,hyaluronic acid is present at about 3 mg/mL to about 100 mg/mL, about 6mg/mL to about 24 mg/mL, about 1 mg/mL to about 30 mg/mL, about 6 mg/mL,about 9 mg/L, about 12 mg/mL, about 15 mg/L, about 16 mg/mL, about 18mg/L, about 21 mg/L, or about 24 mg/mL. In some embodiments, higherhyaluronic acid concentration may lead to higher stiffness and/or moreswelling in the crosslinked macromolecular matrix.

Any type of collagen may be used in the methods and compositionsdescribed herein. In some embodiments, collagen type I, collagen typeIII, collagen type IV, collagen type VI, or a combination thereof, maybe used. In some embodiments, a collagen or a collagen componentcomprises collagen type I or collagen type III.

A collagen may be derived from cell culture, animal tissue, plantderived or recombinant means or recombinant means thereof, and may bederived from human, porcine, or bovine sources. Some embodimentscomprise collagen derived from human fibroblast culture. Someembodiments comprise collagen that has been denatured to gelatin.

Collagen concentration in an aqueous pre-reaction solution or acrosslinking reaction mixture may vary. In some embodiments, collagenmay be present at a concentration of about 1 mg/mL to about 40 mg/mL,about 1 mg/mL to about 15 mg/mL, about 3 mg/mL to about 12 mg/mL, about1.7 mg/mL, about 3 mg/mL, about 6 mg/mL, about 8 mg/mL, or about 12mg/mL.

In some embodiments, the weight ratio of hyaluronic acid to collagen ina aqueous pre-reaction solution or a aqueous pre-reaction solution or acrosslinking reaction mixture (e.g. [wt hyaluronic acid]/[wt collagen])may be about 0.5 to about 10, about 1 to about 7, about 0.5 to about 3,about 1 to about 3, about 1 to about 2, about 1, about 2, about 3, about3.5, about 4, about 5, 5.33, about 6, about 7, or any weight ratio in arange bounded by, and/or between, any of these values. When thecrosslinking reaction occurs, the resulting crosslinked macromolecularproduct may have a collagen component derived from the collagen in thecrosslinking reaction. Thus, the resulting crosslinked macromolecularmatrix may have a weight ratio of hyaluronic acid component to collagencomponent that corresponds to the weight ratio in the crosslinkingreaction, e.g. about 0.5 to about 10, about 1 to about 7, about 0.5 toabout 3, about 1 to about 3, about 1 to about 2, about 1, about 2, about3, about 3.5, about 4, about 5, 5.33, about 6, about 7, or any weightratio in a range bounded by, and/or between, any of these values. Ahigher weight ratio of hyaluronic acid to collagen may result in acrosslinked macromolecular matrix with increased swelling, decreasedstiffness, and/or decreased cell adhesion.

Certain advantageous compositions of the invention include compositionshaving a hyaluronic acid to collagen weight ratio of about 3:3, about12:6, about 16:8, about 12:12, about 12:24, about 12:3, about 16:3, orabout 20:3 (mg/ml).

In some embodiments, the weight ratio of hyaluronic acid to collagen ina aqueous pre-reaction solution or a aqueous pre-reaction solution or acrosslinking reaction mixture may be about 12 mg/mL of hyaluronic acidto about 6 mg/mL collagen, about 12 mg/mL of hyaluronic acid to about 12mg/mL collagen, or about 16 mg/mL of hyaluronic acid to about 8 mg/mLcollagen. In some embodiments, the collagen may be collagen type 1.

An increase in the amount of both hyaluronic acid and collagen mayresult in a crosslinked macromolecular matrix with increased stiffness.

A salt may help to screen the negative charges of hyaluronic acid fromthe positive charges of collagen, and may thus prevent precipitation ofa polyionic ion complex from solution. However, high concentrations ofsalt may reduce the solubility of some components in solution. Thus, insome embodiments, the salt concentration of an aqueous pre-reactionsolution or a crosslinking reaction mixture may be high enough to screenthe charges so that the polyionic ion complex is not formed, but alsolow enough so that the components of the mixture remain in solution. Forexample, the total salt concentration of some aqueous pre-reactionsolutions or crosslinking reaction mixtures may be about 10 mM to about1 M, about 100 mM to about 300 mM, or about 150 mM. In some embodiments,a higher salt concentration may increase the efficiency of acrosslinking reaction, which may result in lower swelling and/or higherstiffness.

Some salts in an aqueous pre-reaction solution or a crosslinkingreaction mixture may be non-coordinating buffers. Any non-coordinatingbuffer may be used that is capable of buffering the mixture and does notform coordinating complexes with coupling agents or metal atoms.Examples of suitable non-coordinating buffers may include, but are notlimited to, 2-(N-morpholino)ethanesulfonic acid (MES),3-(N-morpholino)propanesulfonic acid (MOPS),4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid (HEPES),3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS),N-cyclohexyl-2-aminoethanesulfonic acid (CHES),N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), etc.

The concentration of a non-coordinating buffer may vary. For example,some aqueous pre-reaction solutions or crosslinking reaction mixturesmay have a buffer concentration in a range of about 10 mM to about 1 M,about 10 mM to about 500 mM, about 20 mM to about 100 mM, or about 25 mMto about 250 mM. Some aqueous pre-reaction solutions or crosslinkingreaction mixtures comprise MES at a concentration of about 20 mM toabout 200 mM, about 20 mM to about 100 mM, about 100 mM, or about 180mM.

Non-buffering salts may also be included in an aqueous pre-reactionsolution or a crosslinking reaction mixture as an alternative to, or inaddition, to buffering salts. Some examples may include sodium chloride,potassium chloride, lithium chloride, potassium bromide, sodium bromide,lithium bromide, and the like. The concentration of a non-buffering saltmay vary. For example, some mixtures may have a non-buffering saltconcentration in a range of about 10 mM to about 1 mM, about 30 mM toabout 500 mM, or about 50 mM to about 300 mM. In some embodiments,sodium chloride may be present at a concentration in a range of about0.5% w/v to about 2% about 0.9% w/v, about 1.6% w/v, about 20 mM toabout 1 mM, about 40 mM to about 500 mM, about 50 to 300 mM, about 80 mMto about 330 mM, about 150 mM, or about 270 mM.

The pH of an aqueous pre-reaction solution may be lower than the pH of acrosslinking reaction mixture. If the salt content of the aqueouspre-reaction solution is low, the pH may be lower to enhance solubilityof the hyaluronic acid and the collagen. If the salt content is higher,the pH may be higher in the aqueous pre-reaction solution. In someembodiments, the pH of the aqueous pre-reaction mixture is about 1 toabout 8, about 3 to about 8, about 4 to about 6, about 4.7 to about 7.4,or about 5.4. For low salt concentrations, the pH may be about 1 toabout 4 or about 1 to about 3. In some embodiments, a pH of around 5.4may result in a crosslinked macromolecular matrix having higherstiffness and/or lower swelling.

In some embodiments, pH may be adjusted to neutral to allow collagengelation or fiber formation before adding a coupling agent.

In some embodiments, the pH may be adjusted to neutral immediately priorto, around the time of, or after adding a coupling agent, such thatcollagen gelation is reduced or does not substantially occur.

Any water-soluble coupling agent may be used that can crosslinkhyaluronic acid to collagen. Some non-limiting examples of a couplingagent include carbodiimides such as N,N′-dicyclohexylcarbodiimide (DCC),N,N′-diisopropylcarbodiimide (DIC), or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), etc. Carbodiimidecoupling agents may facilitate ester or amide bond formation withoutbecoming part of the linkage. In other words, an ester bond or an amidebond may comprise atoms from a carboxylate group from one of hyaluronicacid or collagen, and a hydroxyl group or an amine group from the other.However, other coupling agents that become part of the crosslinkinggroup may be used. The concentration of a coupling agent may vary. Insome embodiments, a coupling agent may be present at about 2 mM to about150 mM, about 2 mM to about 50 mM, about 20 mM to about 100 mM, or about50 mM. In some embodiments, the coupling agent is EDC that is present ata concentration of about 20 mM to about 100 mM, about 2 mM to about 50mM, or about 50 mM.

A crosslinking reaction includes any reaction wherein hyaluronic acid iscovalently linked to collagen in a plurality of (e.g. more than 1)positions. In some embodiments, a crosslinking reaction may berepresented by Scheme 1 below. In Scheme 1, only some of the reactingfunctional groups are depicted. Additionally, some functional groupsthat may potentially react in a crosslinking reaction, but may remainunreacted. Unreacted functional groups such as these are not shown. Forexample, OH, CO₂H, —NHCOCH₃, and other groups on hyaluronic acid thatare not shown may react, but may also remain unreacted. Similarly,collagen may have additional groups that may react, but may also remainunreacted, such as OH, SH, CO₂H, NH₂, etc. Additionally, fewer groupsmay react than those depicted.

In Scheme 1, functional groups such as CO₂H on hyaluronic acid may reactwith functional groups on collagen such as NH₂ and OH to form severalcrosslink units. The crosslink units together make up the crosslinkingcomponent. In Scheme 1, a coupling component does not become part of acrosslink unit. However, for some coupling agents, at least part of acoupling agent may be incorporated into a crosslink unit. The hyaluronicacid component includes hyaluronic acid that has reacted to become partof a crosslinked macromolecular matrix. The collagen component includescollagen that has reacted to become part of a crosslinked macromolecularmatrix. In addition to the crosslinking between hyaluronic acid andcollagen, hyaluronic acid or collagen may be partially self-crosslinked.Thus, Scheme 1 is presented for convenience in understanding thecrosslinking reaction, but does not necessarily reflect an actualchemical structure. For example, a crosslinked molecular matrix may be anetwork of hyaluronic acid macromolecules and collagen macromolecules,with many macromolecules crosslinked to more than one macromolecule.

As a result of a crosslinking reaction, a crosslinked macromolecularmatrix may comprise a crosslinking component that crosslinks orcovalently connects the hyaluronic acid component to the collagencomponent. As explained above, a crosslink component comprises aplurality of crosslink units, or individual covalent bonding links,between the hyaluronic acid component and the collagen component. Acrosslink unit may simply be a direct bond between a hyaluronic acidcomponent and a collagen component, so that the coupling agent may notbe incorporated into the crosslinked macromolecular matrix.Alternatively, a crosslink unit may contain additional atoms or groupsfrom the coupling agent such that at least a portion of the couplingagent may become part of the crosslinked macromolecular matrix. At leasta portion of the crosslink units comprise an ester bond or an amidebond. In some embodiments, at least a portion of the crosslink units maybe —CON— or —CO₂—, where the N is a nitrogen from an amino acid residue.

An activating agent may be used to increase the rate of the crosslinkingreaction and the number of crosslink units in the final product. In someembodiments, an activating agent may be a triazole such ashydroxybenzotriazole (HOBT) or 1-hydroxy-7-azabenzotriazole (HOAT); afluorinated phenol such as pentafluorophenol; a succinimide such asN-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sulfoNHS), andthe like.

The concentration of an activating agent may vary. In some embodiments,the activating agent may have a concentration of about 2 mM to about 200mM, about 2 mM to about 50 mM, about 20 mM to about 100 mM, or about 50mM. In some embodiments, the activating agent may be NHS or sulfoNHS isat a concentration of about 2 mM to about 50 mM. In some embodiments,the activating agent may be N-hydroxysulfosuccinimide, sodium salt, at aconcentration of about 20 mM to about 100 mM, or about 50 Mm.

In some embodiments, a crosslinking reaction mixture may comprise acarbodiimide coupling agent and an activating agent. In someembodiments, the coupling agent is EDC and the activating agent is NHSor sulfoNHS. In some embodiments EDC is present at a concentration ofabout 2 mM to about 50 mM and NHS or sulfoNHS is present at about 2 mMto about 50 mM.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 3 mg/mL, human collagen typeIII at a concentration of about 3 mg/mL, 2-(N-morpholino)ethanesulfonicacid at a concentration of about 100 mM, sodium chloride at aconcentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 6 mg/mL, human collagen typeIII at a concentration of about 6 mg/mL, 2-(N-morpholino)ethanesulfonicacid at a concentration of about 180 mM, sodium chloride at aconcentration of about 1.60.9 wt % or about 270 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 16 mg/mL of, rat collagentype I at a concentration of about 8 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 12 mg/mL, rat collagen typeI at a concentration of about 12 mg/mL, 2-(N-morpholino)ethanesulfonicacid at a concentration of about 100 mM, sodium chloride at aconcentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 12 mg/mL, rat tail collagentype I at a concentration of about 12 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.3.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 3 mg/mL, human collagen typeI at a concentration of about 3 mg/mL, 2-(N-morpholino)ethanesulfonicacid at a concentration of about 100 mM, sodium chloride at aconcentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 12 mg/mL, human collagentype I at a concentration of about 6 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 16 mg/mL, human collagentype I at a concentration of about 8 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 12 mg/mL, human collagentype I at a concentration of about 12 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 24 mg/mL, human collagentype I at a concentration of about 12 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 16 mg/mL, human collagentype I at a concentration of about 3 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 9 mg/mL, human collagen typeI at a concentration of about 3 mg/mL, 2-(N-morpholino)ethanesulfonicacid at a concentration of about 100 mM, sodium chloride at aconcentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 12 mg/mL, human collagentype I at a concentration of about 3 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 15 mg/mL, human collagentype I at a concentration of about 3 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 18 mg/mL, human collagentype I at a concentration of about 3 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 21 mg/mL, human collagentype I at a concentration of about 3 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 9 mg/mL, human collagen typeI at a concentration of about 6 mg/mL, 2-(N-morpholino)ethanesulfonicacid at a concentration of about 100 mM, sodium chloride at aconcentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 15 mg/mL, human collagentype I at a concentration of about 6 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 18 mg/mL, human collagentype I at a concentration of about 6 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 21 mg/mL, human collagentype I at a concentration of about 6 mg/mL,2-(N-morpholino)ethanesulfonic acid at a concentration of about 100 mM,sodium chloride at a concentration of about 0.9 wt % or about 150 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 50 mM, and N-hydroxysulfosuccinimide sodium salt at aconcentration of about 50 mM, wherein the solution has a pH of about5.4.

In some embodiments, a crosslinking reaction mixture may comprisehyaluronic acid at a concentration of about 1 mg/mL to about 20 mg/mL,porcine collagen type I at a concentration of about 1 mg/mL to about 15mg/mL, 2-(N-morpholino)ethanesulfonic acid at a concentration of about20 mM to about 200 mM, sodium chloride at a concentration of about 0.5wt % to about 2 wt % or about 80 mM to about 330 mM,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration ofabout 20 mM to about 100 mM, and N-hydroxysulfosuccinimide sodium saltat a concentration of about 20 mM to about 100 mM, wherein the solutionhas a pH of about 4 to about 6.

Aspects of the present specification provide, in part, a compositioncomprising a hydrogel as described herein, and a cellular component. Inan embodiment, a cellular component comprises cells obtained fromadipose tissue. In some embodiments, a cellular component comprisesadipocytes, adipose-derived stem cells, stromal vascular fraction cells,or a combination thereof.

As used herein, the terms “adipose tissue,” “fat,” “fat tissue”, or“fatty tissue” include loose fibrous connective tissue comprising fatcells (adipocytes) and multiple types of regenerative cells, and maycomprise brown and/or white adipose tissue taken from any body site,such as, e.g., subcutaneous, omental/visceral, interscapular, ormediastinal. It may be obtained from any organism having adipose tissue,or the adipose tissue used may be from a primary cell culture or animmortalized cell line.

Adipose tissue may be collected from the same individual who isundergoing the soft tissue replacement procedure (autograft), from adonor individual who is not the same individual as the one undergoingthe soft tissue replacement procedure (allograft), or from an animalsource (xenograft). As used herein, the term “autotransplantation”refers to the transplantation of organs, tissues, or cells from one partof the body to another part in the same individual, i.e., the donor andrecipient are the same individual. Tissue transplanted by such“autologous” procedures is referred to as an autograft orautotransplant. As used herein, the term “allotransplantation” refers tothe transplantation of organs, tissues, or cells from a donor to arecipient, where the donor and recipient are different individuals, butof the same species. Tissue transplanted by such “allologous” proceduresis referred to as an allograft or allotransplant. As used herein, theterm “xenotransplantation” refers to the transplantation of organs,tissues, or cells from a donor to a recipient, where the donor is of adifferent species as the recipient. Tissue transplanted by such“xenologous” procedures is referred to as a xenograft or xenotransplant.

Adipose tissue can be collected by any procedure that can harvestadipose tissue useful for the compositions and methods disclosed herein,including, without limitation a liposuction (lipoplasty) procedure or alipectomy procedure. Procedures useful for collecting adipose tissueshould minimize the trauma and manipulation associated with adiposetissue removed. Adipose tissue may be harvested from any suitableregion, including, without limitation, a mammary region, an abdominalregion, a thigh region, a flank region, a gluteal region, a trochanterregion, or a gonadal region. Procedures useful for collecting adiposetissue are well known to a person of ordinary skill in the art. Theselected procedures may be performed concomitantly with liposculpture.

A liposuction procedure harvests adipose tissue by aspirating the tissueusing a cannula. The cannula may be connected to a syringe for manualaspiration or to a power assisted suction device, like an aspirator,adapted to collect the adipose tissue into a vacuum bottle. Aliposuction procedure does not maintain an intact blood supply of theharvested tissue. The syringe may be a 10, 20 or 60 mL syringe fittedwith a 12 or 14 gauge cannula. Non-limiting examples of liposuctionprocedures include suction-assisted liposuction (SAL),ultrasound-assisted liposuction (UAL), power-assisted liposuction (PAL),twin-cannula (assisted) liposuction (TCAL or TCL), or externalultrasound-assisted liposuction (XUAL or EUAL), or water-assistedliposuction (WAL). In addition, the liposuction procedures listed abovecan be used with any of the following procedures that vary the amount offluid injected during the procedure, such as, e.g., dry liposuction, wetliposuction, super-wet liposuction, tumescent liposuction, orlaser-assisted liposuction. An autologous soft tissue transfer proceduretypically uses adipose tissue collected from a liposuction procedure.

Although the harvested tissue may be used directly to make the disclosedcompositions, it is more typically processed to purify and/or enrich forhealthy adipocytes and regenerative cells. For example, the harvestedadipose tissue may be separated from any debris and/or contaminants suchas, e.g., blood, serum, proteases, lipases, lipids and other oils,and/or other bodily fluids; tumescent fluid and/or other materials usedin the liposuction procedure; and/or other impurities suctioned duringthe procedure. Methods useful in separating debris and/or contaminantsfrom adipose tissue useful to make the disclosed compositions,including, without limitation, centrifugation, sedimentation,filtration, and/or absorption. In addition, or alternatively, theharvested adipose tissue may be processed by washing is a physiologicalbuffer like saline to remove any debris and/or contaminants.

A lipectomy procedure harvests adipose tissue by surgical excision froma donor site in a manner that minimizes damage to the blood supply ofthe tissue using standard surgical operative procedures. This harvestedtissue is then implanted into the region needing the soft tissuereplacement. A tissue flap or tissue graft procedure typically usesadipose tissue collected from a lipectomy procedure. A tissue flap is asection of living tissue that maintained its blood supply as the tissueis moved from one area of the body to another.

A local flap uses a piece of skin and underlying tissue that lieadjacent to the wound, including adipose tissue. The flap remainsattached at one end so that it continues to be nourished by its originalblood supply, and is repositioned over the wounded area. A regional flapuses a section of tissue that is attached by a specific blood vessel.When the flap is lifted, it needs only a very narrow attachment to theoriginal site to receive its nourishing blood supply from the tetheredartery and vein. A musculocutaneous flap, also called a muscle and skinflap, is used when the area to be covered needs more bulk and a morerobust blood supply. Musculocutaneous flaps are often used in breastreconstruction to rebuild a breast after mastectomy. As an example, thetransverse rectus abdominis myocutaneous) flap (TRAM flap) is a tissueflap procedure that uses muscle, fat and skin from an abdomen to createa new breast mound after a mastectomy. This type of flap remains“tethered” to its original blood supply. In a bone/soft tissue flap,bone, along with the overlying skin, is transferred to the wounded area,carrying its own blood supply.

Typically, a wound that is wide and difficult or impossible to closedirectly may be treated with a skin graft. A skin graft is a patch ofhealthy skin that is taken from one area of the body, called the “donorsite,” and used to cover another area where skin is missing or damaged.There are three basic types of skin grafts. A split-thickness skingraft, commonly used to treat burn wounds, uses only the layers of skinclosest to the surface. A full-thickness skin graft might be used totreat a burn wound that is deep and large, or to cover jointed areaswhere maximum skin elasticity and movement are desired. As its nameimplies, a full-thickness (all layers) section of skin from the donorsite are lifted. A composite graft is used when the wound to be coveredneeds more underlying support, as with skin cancer on the nose. Acomposite graft requires lifting all the layers of skin, adipose tissue,and sometimes the underlying cartilage from the donor site.

The amount of adipose tissue collected will typically vary fromindividual to individual and can depend on a number of factorsincluding, but not limited to, amount of adipose tissue required for thesoft tissue replacement method, aesthetic expectations, age, bodyhabitus, coagulation profile, hemodynamic stability, co-morbidities, andphysician preference. A liposuction procedure may harvest from about 1mL to about 1500 mL of adipose tissue. A lipectomy procedure typicallyharvests about 1 g to about 5,000 g.

Adipose tissue comprises multiple types of regenerative cells. As usedherein, the term “regenerative cell” refers to any cells that cause orcontribute to complete or partial regeneration, restoration, orsubstitution of structure or function of an organ, tissue, orphysiologic unit or system to thereby provide a therapeutic, structuralor cosmetic benefit. Examples of regenerative cells include stem cells,progenitor cells, and precursor cells.

As used herein, the term “stem cell” refers to a multipotentregenerative cell with the potential to differentiate into a variety ofother cell types that perform one or more specific functions and has theability to self-renew. Some of the stem cells disclosed herein may bepluripotent. Exemplary examples of stem cells include, withoutlimitation, adipose-derived stem cells (ASCs; adipose-derived stromalcells), endothelial-derived stem cells (ESCs), hemopoietic stem cells(HSCs), and mesenchymal stem cells (MSCs). Examples of differentiationinclude angiogenesis, neovascularization, adipogenesis and collagenesis.

As used herein, the term “progenitor cell” refers to an oligopotentregenerative cell with the potential to differentiate into more than onecell type, or a unipotent regenerative cell with the potential todifferentiate into only a single cell type, that perform(s) one or morespecific functions and has limited or no ability to self-renew.Exemplary examples of progenitor cells include, without limitation,endothelial progenitor cells, keratinocytes, monoblasts, myoblasts, andpericytes.

As used herein, the term “precursor cell” refers to a unipotentregenerative cell with the potential to differentiate into one cell typethat performs one or more specific functions and may retain extensiveproliferative capacity that enables the cells to proliferate underappropriate conditions. Exemplary examples of precursor cells include,without limitation, adipoblast (lipoblast or preadipocytes),de-differentiated adipocytes, angioblasts, endothelial precursor cells,fibroblasts, lymphoblasts, and macrophages.

A hydrogel composition disclosed herein may enhance differentiation ofthe multiple regenerative cells from the adipose tissue. In oneembodiment, a hydrogel composition disclosed herein enhancesdifferentiation of the multiple regenerative cells from the adiposetissue as compared to adipose tissue alone. In aspects of thisembodiment, a hydrogel composition disclosed herein enhancesdifferentiation of the multiple regenerative cells from the adiposetissue by at least about 50% at least about 100%, at least about 150%,at least about 200%, at least 250%, at least about 300%, at least about350%, at least about 400%, at least about 450%, at least about 500%, atleast about 750%, or at least about 1000% as compared to adipose tissuealone. In aspects of this embodiment, a hydrogel composition disclosedherein enhances differentiation of the multiple regenerative cells fromthe adipose tissue by about 50% to about 250%, about 50% to about 500%,about 50% to about 1000%, about 100% to about 300%, about 100% to about500%, about 100% to about 1000%, about 150% to about 400%, about 150% toabout 600%, about 150% to about 1000%, about 200% to about 500%, about200% to about 700%, or about 200% to about 1000% as compared to adiposetissue alone.

In another embodiment, a hydrogel composition disclosed herein enhancesdifferentiation of the multiple regenerative cells from the adiposetissue as compared to adipose tissue with a hydrogel composition that issubstantially identical except that the hyaluronic acid component andthe collagen component are not crosslinked. In aspects of thisembodiment, a hydrogel composition disclosed herein enhancesdifferentiation of the multiple regenerative cells from the adiposetissue by at least about 50% at least about 100%, at least about 150%,at least about 200%, at least 250%, at least about 300%, at least about350%, at least about 400%, at least about 450%, at least about 500%, atleast about 750%, or at least about 1000% as compared to adipose tissuewith a hydrogel composition that is substantially identical except thatthe hyaluronic acid component and the collagen component are notcrosslinked. In aspects of this embodiment, a hydrogel compositiondisclosed herein enhances differentiation of the multiple regenerativecells from the adipose tissue by about 50% to about 250%, about 50% toabout 500%, about 50% to about 1000%, about 100% to about 300%, about100% to about 500%, about 100% to about 1000%, about 150% to about 400%,about 150% to about 600%, about 150% to about 1000%, about 200% to about500%, about 200% to about 700%, or about 200% to about 1000% as comparedto adipose tissue with a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the collagencomponent are not crosslinked. In another aspect of this embodiment, ahydrogel composition that is substantially identical to a hydrogelcomposition disclosed herein except that the hyaluronic acid componentand the collagen component are not crosslinked comprises hyaluronic acidat a concentration of about 16 mg/mL or about 24 mg/mL and water.

In yet another embodiment, a hydrogel composition disclosed hereinenhances differentiation of the multiple regenerative cells from theadipose tissue as compared to adipose tissue with a hydrogel compositionthat is substantially identical except that the collagen component isabsent. In aspects of this embodiment, a hydrogel composition disclosedherein enhances differentiation of the multiple regenerative cells fromthe adipose tissue by at least about 50% at least about 100%, at leastabout 150%, at least about 200%, at least 250%, at least about 300%, atleast about 350%, at least about 400%, at least about 450%, at leastabout 500%, at least about 750%, or at least about 1000% as compared toadipose tissue with a hydrogel composition that is substantiallyidentical except that the collagen component is absent. In aspects ofthis embodiment, a hydrogel composition disclosed herein enhancesdifferentiation of the multiple regenerative cells from the adiposetissue by about 50% to about 250%, about 50% to about 500%, about 50% toabout 1000%, about 100% to about 300%, about 100% to about 500%, about100% to about 1000%, about 150% to about 400%, about 150% to about 600%,about 150% to about 1000%, about 200% to about 500%, about 200% to about700%, or about 200% to about 1000% as compared to adipose tissue with ahydrogel composition that is substantially identical except that thecollagen component is absent. In another aspect of this embodiment, ahydrogel composition that is substantially identical to a hydrogelcomposition disclosed herein except that the collagen component isabsent comprises hyaluronic acid at a concentration of about 16 mg/mL orabout 24 mg/mL and water.

Harvested adipose tissue useful in compositions of the invention can besupplemented with regenerative cells such as, e.g., stem cells,progenitor cells, and precursor cells. Regenerative cells may promotenew blood vessel formation, diminish necrosis, and/or promote asupportive microenvironment in the transplanted tissue, therebyimproving survivability of the transplanted tissue. Regenerative cellscan be obtained from a variety of sources. For example, adipose tissueis rich in regenerative cells that have the ability to restore andreconstruct various soft tissue defects in response to localdifferentiation clues from the recipient site. As such, a portion of thecollected adipose tissue may be further processed in order to purifyregenerative cells that can then be added back to the remainder of theharvested adipose tissue in order to enrich this material for thesecells. Exemplary methods describing such cell enrichment procedures canbe found in, e.g., Hedrick and Fraser, Methods of Using AdiposeTissue-Derived Cells in Augmenting Autologous Fat Transfer, U.S. PatentPublication 2005/0025755, Yoshimura, et al., Characterization of FreshlyIsolated and Cultured Cells Derived form the Fatty and Fluid Portions ofliposuction Aspirates, J. Cell. Physiol. 208: 1011-1041 (2006);Yoshimura, et al., Cell-Assisted Lipotransfer for Facial Lipoatrophy:Effects of Clinical Use of Adipose-Derived Stem Cells, Dermatol. Surg.34: 1178-1185 (2008); Yoshimura, et al., Cell-Assisted Lipotransfer forCosmetic Breast Augmentation: Supportive Use of Adipose-DerivedStem/Stromal Cells, Aesth. Plast. Surg. 32: 48-55 (2008); each of whichis hereby incorporated by reference in its entirety.

In addition, harvested adipose tissue can be supplemented withregenerative cells obtained from cell cultures, such as, e.g., primarycell cultures and established cell cultures. For example, a portion ofharvested adipose tissue from an individual can be cultured in a mannerto produce primary cell cultures enriched for regenerative cells.Alternatively, established cell lines derived from regenerative cellsfrom adipose tissue, or another tissue source, can be cultured,harvested, and added to adipose tissue collected form an individual.Exemplary methods describing such cell culture compositions andprocedures can be found in, e.g., Casteilla, et al., Method forCulturing Cells Derived from the Adipose Tissue and Uses Thereof, U.S.Patent Publication 2009/0246182; Chazenbalk, et al, Methods of ProducingPreadipocytes and Increasing the Proliferation of Adult AdiposeStem/Progenitor Cells, U.S. Patent Publication 2009/0317367; Kleinsekand Soto, Augmentation and Repair of Sphincter Defects with CellsIncluding Adipocytic Cells, U.S. Patent Publication 2008/0299213;Rehman, et al., Secretion of Angiogenic and Antiapoptotic Factors byHuman Adipose Stromal Cells, Circulation 109: r52-r58 (2004); Kilroy, etal., Cytokine Profile of Human Adipose-Derived Stem Cells: Expression ofAngiogenic, Hematopoietic, and Pro-Inflammatory Factors, J. Cell.Physiol. 212: 702-709 (2007); each of which is hereby incorporated byreference in its entirety.

Harvested adipose tissue may be immediately used to make thecompositions disclosed herein. Alternatively, harvested adipose tissue,whether unprocessed or processed, may be stored for used at some futuredate. Harvested tissue is typically stored using a slow freezing methodof the tissue to −20° C., with or without cryopreservatives. Storedadipose tissue can typically be stored for at least 6 months.

In some embodiments, a hydrogel composition as described herein mayinclude a hyaluronic acid:collagen weight ratio of 3 to 1. Theconcentrations of hyaluronic acid can be from about 12 mg/mL to about 24mg/mL and the collagen can be from about 3 mg/mL to about 12 mg/mL. Thecollagen may be collagen type 1. Further, the hydrogel composition maybe used for fat grafting applications as an additive. The source of thecollagen can vary, but can be human recombinant (cell derived or plantderived), porcine, bovine or ovine. The hydrogels can be formed with anEDC crosslinker and NHS as an activating agent.

Hydrogel compositions described herein can further have a storagemodulus (G′) and a loss modulus (G″) each independently between about500 Pa and about 4,000 Pa.

A general method of making hydrogel compositions as described herein canbe achieved as follows. First, lyophilized hyaluronic acid fibers can beadded to a concentrated (e.g. hydrated) collagen solution. The pH canthen be managed by the addition of one or more buffer salt and/or theaddition of a base (e.g. NaOH). After the pH has been managed, themixture can be hydrated and thoroughly mixed followed by addition ofcrosslinking agents. The crosslinking agents can be solids (e.g.powder). The hyaluronic acid and collagen can be left to react. Oncereacted, the resultant gel can be particle sized through a filter mesh(e.g. 100 μm) and can be dialyzed with buffer to purify (e.g. againstany unused or unreacted crosslinker). The gel can then be sterilized(e.g. using isopropanol). This sterilization can also occur prior topurification. Once sterilized the gel may be ready for administration.The sterilized gel can also be further mixed within adipose tissue (e.g.human).

The sterilized gel either mixed with adipose tissue or not mixed withadipose tissue can be administered as described herein to treat acondition of, for example, the face, breast, hands, etc.

Example 1

Hyaluronic acid, 2 MDa molecular weight, (HTL Biotech) was dissolved inhuman collagen(I) solution in 0.01 N hydrochloric acid (AdvancedBioMatrix). Sodium chloride was added at 0.9 wt % and2-(morpholino)ethanesulfonic acid was added at 100 mM to the solutionand mixed. The hyaluronic acid was allowed to hydrate for 1 hr and thesolution was homogenized by syringe-to-syringe mixing. The pH of thesolution was adjusted to 5.4 by addition of 1 N sodium hydroxide.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (50 mM) andN-hydroxysulfosuccinimide sodium salt (50 mM) were added to thehyaluronic acid/collagen solution and quickly mixed bysyringe-to-syringe transfer. The solution was transferred to a glassvial and centrifuged for 5 min at 4000 RPM to remove air bubbles. Theresulting gel was allowed to react for 16 hrs at 4° C. The gel was thenparticulated through a 100 micron pore-sized mesh. Following sizing, thegel was sterilized by dialysis through a 20 kDa molecular-weight cut-offcellulose ester membrane against 70% isopropanol/30% water for 3 hrs at4° C. Dialysis was then continued against sterile phosphate buffer, pH7.4, for 48 hrs at 4° C. with four changes of buffer. The gel was thendispensed into syringes under aseptic conditions.

This procedure was used to produce hydrogels with varying concentrationsof hyaluronic acid and collagen. When required, human collagen(I) in0.01 N hydrochloric acid was concentrated from 3 mg/mL to the desiredreaction concentration in 20 kDa molecular-weight cut-off centrifugalfiltration devices. A 50 mL sample of each gel was synthesized,sterilized by exposure to 70% isopropanol, and purified by dialysisagainst phosphate buffer, pH 7.4. The gels synthesized are described inTable 1.

TABLE 1 Hyaluronic acid-human collagen(I) hydrogel synthesisconcentrations Sample [HA] [Col(I)] ID (mg/mL) (mg/mL) A 3 3 B 12 6 C 168 D 12 12 E 24 12

Example 2

Oscillatory parallel plate rheology was used to characterize themechanical properties of the hydrogels synthesized in Example 1 using anAnton Paar MCR 301. A plate diameter of 25 mm was used at a gap heightof 1 mm. A frequency sweep from 0.1 to 10 Hz at a fixed strain of 2%with logarithmic increase in frequency was applied followed by a strainsweep between 0.1% and 300% at a fixed frequency of 5 Hz withlogarithmic increase in strain. The storage modulus (G′) and lossmodulus (G″) were recorded from frequency sweep measurements at 5 Hz.Values from measurements of samples from Example 1 are presented inTable 2.

TABLE 2 Rheological properties of hyaluronic acid-collagen(I) hydrogelsSample [HA] [Col(I)] G′ G″ ID mg/mL) (mg/mL) (Pa) (Pa) A 3 3 199 24.6 B12 6 1260 154 C 16 8 2450 288 D 12 12 3160 420 E 24 12 5440 433 F 12 31100 52.2 G 16 3 1490 60.6 H 20 3 1770 49.5

Example 3

Swelling ratios were determined relative to initial water content foreach of the samples in Example 1 by increase in weight when equilibratedwith phosphate buffer. For each gel, approximately 1 mL was injectedinto a 15 mL Falcon tube and weighed followed by addition of 10 mL ofphosphate buffered saline, pH 7.4. The gels were thoroughly mixed withthe buffer and vortexed for 30 seconds. The gels were then allowed toequilibrate in the buffer for 48 hrs at 4° C. After this time, thesuspensions were centrifuged at 4000 RPM in a swinging bucket rotor for5 minutes. The supernatant buffer was then decanted and the weight ofthe swollen gel was measured. The swelling ratio was determined bydividing the final weight of the swollen gel by the weight of theinitial gel. The swelling results of samples from Example 1 arepresented in Table 3. A swelling ratio less than 1 indicates that thegel lost water upon equilibration and centrifugation.

TABLE 3 Swelling ratios of hyaluronic acid-collagen(I) hydrogels Sample[HA] [Col(I)] Swelling ID (mg/mL) (mg/mL) ratio A 3 3 0.96 B 12 6 1.67 C16 8 1.69 D 12 12 1.49 E 24 12 1.65

Example 4

Samples of gels from Example 1 were tested for their ability to supporthuman adipose derived stem cell (ASC) viability. In 96-well plates, 50μL gel beds were created in triplicate from gels of Example 1.Culture-expanded ASCs (Invitrogen) were plated at 5,000 cells/cm² on thegel beds in MesenPro RS medium with growth supplement (Invitrogen, CA).The cells were cultured for 18 hrs at 37° C., 5% CO₂, after which theMTT assay (ATCC, VA) was performed. The tetrazolium compound MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) is added tothe wells and the cells are incubated. MTT is reduced by metabolicallyactive cells to insoluble purple formazan dye crystals. Detergent isthen added to the wells, solubilizing the crystals so the absorbance canbe read using a spectrophotometer at 570 nm, Cells adhered to the gelsand exhibited a spread morphology. The viability of hASCs increased withincreasing total biopolymer and collagen concentrations. Viabilityrelative to tissue culture polystyrene (TCP) are shown in Table 4.

TABLE 4 ASC viability on hyaluronic acid-collagen(I) hydrogels [HA][Col(I)] Viability Sample (mg/mL) (mg/mL) (relative to TCP) A 3 3  94% B12 6 120% C 16 8 140% D 12 12 245% E 24 12 350%

Example 5

Samples of HA-Col(I) from Example 1 were tested for their ability tosupport ASC proliferation. In 96-well plates, 50 μL gel beds werecreated in triplicate from the hyaluronic acid-collagen(I) hydrogels.ASCs (Invitrogen) were plated at 5,000 cells/cm² on the gel beds inMesenPro RS medium with growth supplement (Invitrogen). ASCs werecultured for one week at 37° C., 5% CO₂, and proliferation wasdetermined by MTT assay at 3, 5, and 7 days with media changes every 2days. Tissue culture polystyrene (TCP) was used as positive control,while crosslinked HA, not containing collagen, was used as an additionalcontrol to establish the effect of collagen in HA-Col(I) formulations.Proliferation rates at day 3, relative to HA and TCP, were determined.Incorporation of collagen I (at concentration ≥6 mg/mL) dramaticallyimproved HA gel's support for hASC proliferation, reaching proliferationrates comparable to TCP positive control (Table 5).

TABLE 5 hASC proliferation (3-day) on hyaluronic acid-collagen(I)hydrogels [HA] [Col(I)] Proliferation (%) Proliferation (%) Example(mg/mL) (mg/mL) (relative to HA) (relative to TCP) A 3 3 371 ± 63  95 ±143 B 12 6 354 ± 60  91 ± 15 C 16 8 460 ± 60 123 ± 45 D 12 12  381 ± 11995 ± 7 E 24 12 415 ± 91 106 ± 12

Example 6

Samples of hyaluronic acid-collagen(I) matrices from Example 1 wereassessed for their ability to allow diffusion of pro-angiogenic(vascular endothelial growth factor, VEGF) and adipose tissue-specificgrowth factors (adiponectin, leptin). Improved diffusion to any or allof these growth factors would support the enhanced survival ofco-grafted tissue, especially fat, since nutrient diffusion may beimportant for sustained tissue viability. To do this, 100 μL of eachhydrogel tested was loaded into a 8 μm transwell (24 well plate) inorder to make a gel column. Known concentrations of target factors wereloaded on top of the gel, diluted in fibroblast basal medium(Cat#PCS-201-030, ATCC). Plates were allowed to incubate at 37° C. with5% CO₂ in a tissue culture incubator, for 60 hours, thereby allowing thefactors to diffuse through the gels. Diffusion of the specified factorsthrough each hydrogel was measured by ELISA in the supernatant in thebottom chamber of the wells. Results indicated improved diffusion of thetargeted factors compared to the reference, crosslinked HA hydrogel notcontaining collagen (Table 6).

TABLE 6 Hyaluronic acid-human collagen (I) hydrogels showed improveddiffusion of adipokines Adiponectin Leptin VEGF Sample (%) (%) (%) A 250± 25 263 ± 1  225 ± 28 B 84 ± 9 171 ± 11 147 ± 6  C 85 ± 4 196 ± 43 155± 33 D 92 ± 9 152 ± 4  127 ± 5  E 130 ± 79 155 ± 19 111 ± 15 Note:diffusion compared to the reference HA gel, lacking collagen type 1.

Example 7

The hyaluronic acid-collagen(I) gels from Example 1 were mixed withhuman lipoaspirate at 2:1 lipo:gel ratio in a nude mouse model to assessthe gels' ability to enhance fat graft viability and volume retention.Human lipoaspirate tissue was procured through means of ultrasound- orsuction-assisted liposuction under informed consent, then consecutivelycentrifuged and washed 3× at 30 g for 5 min, in 1× phosphate bufferedsaline without cations (PBS, Invitrogen) inside a sterile biosafetycabinet. Next, 10 mL of washed lipoaspirate was transferred to a clean100 mL sterile reservoir. To this tissue, 5 mL of sterile hydrogel wasadded and carefully blended by hand using a sterile spatula. The mixingprocedure required 5 to 10 minutes of constant stirring with mechanicaldisruption of large pieces of tissue to generate a homogenous mixture.Then 1 mL syringes were filled with lipoaspirate/hydrogel until theplunger reached the 1 mL mark. The syringe was then capped with asterile female luer-lock cap and maintained on ice blocks until use.Lipoaspirate/hydrogel mixes were implanted as 1 mL bolus subcutaneouslyon the dorsum of female 6-week-old nude mice under anesthesia with twoinjections per mouse. Each gel/lipo mixture was implanted through asmall incision by 16 G cannula and the incision closed using surgicalglue. A total of 14 injections of each material were made. Syringes wereweighed before and after injection to determine the weight of injectedmaterial. After 6 weeks, the gels were harvested and weight and volume(using liquid displacement) were determined for each sample. Sampleswere also processed for histology by H&E staining.

Example 8 Hyaluronic Acid-Human Collagen(I) Hydrogel Compositions forFat Grafting

Hyaluronic acid, 2 MDa molecular weight, (HTL Biotech) was dissolved inhuman collagen(I) solution in 0.01 N hydrochloric acid (AdvancedBioMatrix). Sodium chloride was added at 0.9 wt % and2-(morpholino)ethanesulfonic acid was added at 100 mM to the solutionand mixed. The hyaluronic acid was allowed to hydrate for 1 hr and thesolution was homogenized by syringe-to-syringe mixing. The pH of thesolution was adjusted to 5.4 by addition of 1 N sodium hydroxide.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (50 mM) andN-hydroxysulfosuccinimide sodium salt (50 mM) were added to thehyaluronic acid/collagen solution and quickly mixed bysyringe-to-syringe transfer. The solution was transferred to a glassvial and centrifuged for 5 min at 4000 RPM to remove air bubbles. Theresulting gel was allowed to react for 16 hrs at 4° C. The gel was thenparticulated through a 100 micron pore-sized mesh. Following sizing, thegel was sterilized by dialysis through a 20 kDa molecular-weight cut-offcellulose ester membrane against 70% isopropanol/30% water for 3 hrs at4° C. Dialysis was then continued against sterile phosphate buffer, pH7.4, for 48 hrs at 4° C. with four changes of buffer. The gel was thendispensed into syringes under aseptic conditions.

This procedure was used to produce hydrogels with varying concentrationsof hyaluronic acid and collagen. When required, human collagen(I) in0.01 N hydrochloric acid was concentrated from 3 mg/mL to the desiredreaction concentration in 20 kDa molecular-weight cut-off centrifugalfiltration devices. A 50 mL sample of each gel was synthesized,sterilized by exposure to 70% isopropanol, and purified by dialysisagainst phosphate buffer, pH 7.4. The gels synthesized are described inTable 7.

TABLE 7 Hyaluronic acid-human collagen(I) hydrogel synthesisconcentrations Sample [HA] [Col(I)] ID (mg/mL) (mg/mL) A 3 3 B 12 6 C 168 D 12 12 E 16 3 F 24 12 G 21 6

Example 9 Hyaluronic Acid-Human Collagen(I) Hydrogels Stimulate HumanAdipose Derived Stem Cell (hASC) Attachment and Proliferation

Samples of HA-Col(I) were tested for their ability to support hASCattachment and proliferation. To evaluate cell attachment, hASCs werecultured on hydrogels in serum free, basal medium for 18 hrs. A sign ofcell attachment is demonstrated in the cell's ability to extendprocesses through the gel. Bright field microscopy demonstrated at 18hrs that hASCs attached and spread on HA-Col(I) hydrogels, but not on HAgels without human collagen(I) (FIGS. 1A and 1B). Actin-phalloidinstaining, to observe individual actin filaments in fixed cultures,further confirmed this result showing clear spreading of cells inHA-Col(I) gels only (data not shown).

To evaluate cell proliferation, in 96-well plates, 50 μL gel beds werecreated in triplicate from the hyaluronic acid-collagen(I) hydrogels.hASCs (Invitrogen) were plated at 5,000 cells/cm² on the gel beds inMesenPro RS medium with growth supplement (Invitrogen). ASCs werecultured for one week at 37° C., 5% CO₂, and proliferation wasdetermined by MTT assay at 3, 5, and 7 days with media changes every 2days. Tissue culture polystyrene (TCP) was used as positive control,while crosslinked hyaluronic acid (HA) not containing collagen, was usedas an additional control to establish the effect of collagen inHA-Col(I) formulations. Proliferation rates at day 5, relative to HA(-)gels not containing collagen, were determined. Incorporation of collagenI significantly improved HA gel's support for hASC proliferation (Table8).

TABLE 8 hASC proliferation (5-day) on hyaluronic acid-collagen(I)hydrogels HA Col1 Percent to HA Sample (mg/mL) (mg/mL) (−) (%) HA(−)[control] 16 0 100 ± 5  HA-CN (9.3) 9 3 337 ± 24 HA-CN (12.3) 12 3 254 ±28 HA-CN (15.3) 15 3 307 ± 22 HA-CN (18.3) 18 3 289 ± 26 HA-CN (21.3) 213 231 ± 46 HA-CN (9.6) 9 6 341 ± 28 HA-CN (12.6) 12 6 501 ± 53 HA-CN(15.6) 15 6 317 ± 34 HA-CN (18.6) 18 6 306 ± 39 HA-CN (21.6) 21 6 300 ±52

Example 10 Hyaluronic Acid-Collagen(I) Hydrogels Support HumanEndothelial and Fibroblast Cell Attachment and Outgrowth

Samples of hyaluronic acid-collagen(I) matrices were tested for theirability to support cell attachment and outgrowth from rat aortic ring.Rat aorta were harvested and dissected into 2 mm×2 mm pieces andembedded in 50 μL gel in 96-well plates. The organ was cultured inendothelial cell growth medium with growth supplement (Lonza). Five dayspost culture, the culture was stained with live cell staining (CalceinAM). Few cells migrated out from HA(-) cultured rat aorta ring, howevermassive cell outgrowth of endothelial and fibroblasts occurred incollagen gel culture (FIG. 2).

Example 11 Hyaluronic Acid-Collagen(I) Hydrogels Support HumanEndothelial Cell Proliferation

Samples of hyaluronic acid-collagen(I) matrices were tested for theirability to support human umbilical vascular endothelial cell (HUVECs)proliferation. In 96-well plates, 50 μL gel beds were created intriplicate from the hyaluronic acid-collagen(I) hydrogels.Culture-expanded HUVECs (Lonza) were plated at 50,000 cells/cm² on thegel beds in endothelial cell growth medium with growth supplement(Lonza). The cells were cultured for up to 7 days at 37° C., 5% CO₂,after which live cell staining (Calcein AM) was performed. HUVECsproliferated very well on hyaluronic acid-collagen gels, but poorly onHA (-) gels. HUVECS attached to and propagated on hyaluronicacid-collagen gels (FIG. 3B), while on HA(-) gels, they did not attach,instead forming cellular aggregates (FIG. 3A).

Example 12 Enhanced Diffusion of Adipose Tissue-Specific andPro-Angiogenic Growth Factors in Hyaluronic Acid-Human Collagen(I)Hydrogels

Samples of hyaluronic acid-collagen(I) matrices from Example 8 wereassessed for their ability to allow diffusion of pro-angiogenic(vascular endothelial growth factor, VEGF) and adipose tissue-specificgrowth factors (adiponectin, leptin). Improved diffusion to any or allof these growth factors would support the enhanced survival ofco-grafted tissue, especially fat, since nutrient diffusion is acritical element to sustained tissue viability. To do this, 100 μL ofeach hydrogel tested was loaded into a 8 μm transwell (24 well plate) inorder to make a gel column. Known concentrations of target factors wereloaded on top of the gel, diluted in fibroblast basal medium(Cat#PCS-201-030, ATCC). Plates were allowed to incubate at 37° C. with5% CO₂ in a tissue culture incubator, for 60 hours, thereby allowing thefactors to diffuse through the gels. Diffusion of the specified factorsthrough each hydrogel was measured by ELISA using the supernatant in thebottom chamber of the wells. Results indicated improved diffusion of thetargeted factors compared to the reference, crosslinked hyaluronic acid(HA) hydrogel not containing collagen (Table 9).

TABLE 9 Hyaluronic acid-human collagen(I) hydrogels exhibit improveddiffusion of adipokines Adiponectin Leptin VEGF Gel (%) (%) (%) HA(−)[control] 100 ± 31  100 ± 21 100 ± 23 HA-CN (16.8) 200 ± 174 167 ± 28119 ± 34 HA-CN (3.3) 211 ± 113 228 ± 6  178 ± 32 HA-CN (12.6) 106 ± 15 157 ± 14 119 ± 15 HA-CN (12.12) 108 ± 15  136 ± 4  103 ± 13 HA-CN(24.12) 189 ± 143 157 ± 54 112 ± 33 Note: diffusion compared to thereference HA gel, lacking collagen type 1 [HA(−)].

Example 13 Hyaluronic Acid-Human Collagen(I) Hydrogels Stimulate HumanAdipose Derived Stem Cell (hASCs) Secretion of Adipokines

Samples of hyaluronic acid matrices with and without human collagen(I)were assayed for cellular leptin secretion following culture with hASCs.Leptin was used as a measure of differentiation into adipocytes in thesethree-dimensional cultures. For each sample, one million hASCs wereencapsulated in 50 μL of hydrogel and cultured in a 0.4 μm transwell ina 24 well tissue culture plate. Cells were cultured using the StemProAdipogenesis Differentiation Kit (Cat#A10070-01, Invitrogen) and wereincubated at 37° C. with 5% CO₂ in a tissue culture incubator. Leptinlevels in the media were measured by ELISA at 7, 14 and 21 days. Theresults in Table 10 indicate that hASCs cultured in HA-collagen(I) gels[HA-CN (16.8) and HA-CN (24.12)] released more leptin than cultures inHA gels lacking collagen(I) [HA(-).]

TABLE 10 Hyaluronic acid-human collagen(I) hydrogels stimulate leptinsecretion in 3D culture. Sample Day 7-Leptin Day 14-Leptin Day 21-LeptinID (ng/mL) (ng/mL) (ng/mL) HA-CN 1.144 ± 0.057 1.235 ± 0.020 1.270 ±0.006 (16.8) HA-CN 1.057 ± 0.099 1.238 ± 0.019 1.262 ± 0.001 (24.12)HA(−) 0.116 ± 0.013 0.261 ± 0.116 0.388 ± 0.046

Example 14 Hyaluronic Acid-Collagen(I) Hydrogels Improve Fat GraftVolume Retention

Samples of hyaluronic acid-collagen(I) from Example 8 were blended withhuman lipoaspirate (lipo) in a volume ratio of 2 parts lipo to 1 partgel, creating a lipo/gel graft. Control, lipo alone samples were usedwith the same lipo tissue volume as lipo/gel blends (0.66 mL). Lipo/gelgrafts or lipo alone controls were implanted in a nude mouse model forevaluation of graft retention, gross tissue morphology, histologic crosssections and biomarkers for adipose tissue. Grafts were implanted as abolus subcutaneously on the dorsum of female 6-week-old nude mice underanesthesia with two injections per mouse. One mL of each mixture wasimplanted through a small incision by 16 G cannula and the incisionclosed using surgical glue. A total of 14 injections of each materialwere made. Syringes were weighed before and after injection to determinethe weight of injected material. After 6 weeks, the grafts wereharvested for end-point assessment. Blending lipoaspirate with severalformulations of hyaluronic acid-collagen(I) from Example 8 improvedlipo/gel graft volume retention above lipoaspirate controls, in all, butone sample (A) (Table 11).

TABLE 11 Lipo/gel volume retention improvement compared to lipoaspiratestudy control at 6 weeks (difference in group means relative to lipoonly control). Improvement in Sample [HA] [Col(I)] Graft Volume ID(mg/mL) (mg/mL) (%) A 3 3 −17 B 12 6 34 C 12 12 32 D 16 3 19 E 16 8 37 F24 12 7 G 21 6 25

Moreover, lipo/gel grafts consistently showed improvements in volumeretention across unique tissue donors and between distinct hydrogellots. Four lots of 12 mg/mL hyaluronic acid-6 mg/mL collagen(I) wereeach blended with lipoaspirate tissue and showed improvements in graftretention ranging from 11% to 34% above lipoaspirate controls (Table12).

TABLE 12 Improvement of lipoaspirate graft percent volume for four 12mg/mL hyaluronic acid - 6 mg/mL collagen(I) unique lots of material.Improvement in Tissue Graft Volume Lot # Donor (%) 0131 A 11 0727 B 150811 C 22 0708 D 34

Example 15 Hyaluronic Acid-Collagen(I) Hydrogels Reduce the Variabilityin Retained Fat Graft Volume

Samples of hyaluronic acid-collagen(I) from Example 8 were blended withhuman lipoaspirate (lipo) in a volume ratio of 2 parts lipo to 1 partgel, creating a lipo/gel graft. Control, lipo alone samples were usedwith the same lipo tissue volume as lipo/gel blends (0.66 mL). Lipo/gelgrafts or lipo alone controls were implanted in a nude mouse model forevaluation of graft retention, gross tissue morphology, histologic crosssections and biomarkers for adipose tissue. Grafts were implanted as abolus subcutaneously on the dorsum of female 6-week-old nude mice underanesthesia with two injections per mouse. One mL of each mixture wasimplanted through a small incision by 16 G cannula and the incisionclosed using surgical glue. A total of 14 injections of each materialwere made. Syringes were weighed before and after injection to determinethe weight of injected material. After 6 weeks, the grafts wereharvested for end-point assessment. Blending hyaluronic acid-collagen(I)with lipoaspirate reduced the variability in graft retention indicatedby a reduction in the mean standard deviation compared to standarddeviation of lipoaspirate controls (Table 13).

TABLE 13 Lipo/gel volume standard deviation compared to lipoaspiratestudy control at 6 weeks (difference in group deviation relative to lipoonly control). Reduction in Sample [HA] [Col(I)] Graft Variability ID(mg/mL) (mg/mL) (%) A 3 3 0 B 12 6 −14 C 12 12 −17 D 16 3 −4 E 16 8 −16F 24 12 0 G 21 6 −4

Example 16 Hyaluronic Acid-Collagen(I) Hydrogels Improve Fat GraftSurvival And Tissue Morphology

Samples of hyaluronic acid-collagen(I) from Example 8 were blended withhuman lipoaspirate (lipo) in a volume ratio of 2 parts lipo to 1 partgel, creating a lipo/gel graft. Lipo/gel grafts were implanted in a nudemouse model for evaluation of graft retention, gross tissue morphology,histologic cross sections and biomarkers for adipose tissue. Grafts wereimplanted as a bolus subcutaneously on the dorsum of female 6-week-oldnude mice under anesthesia with two injections per mouse. One mL of eachmixture was implanted through a small incision by 16 G cannula and theincision closed using surgical glue. A total of 14 injections of eachmaterial were made. Syringes were weighed before and after injection todetermine the weight of injected material. After 6 weeks, the graftswere harvested for histologic end-point assessment. Blending hyaluronicacid-collagen(I) with lipoaspirate improved fat graft survival andtissue morphology as represented through hematoxylin & eosin staining ofparaffin embedded, formalin fixed sections cut throughout the fat/gelgraft (FIGS. 4A-4D).

Turning to FIGS. 4A-4D, photographs of lipo/gel explants as provided asinsets along with corresponding H&E micrographs. Gross examination ofexplanted gel samples indicated signs of reduced fibrosis and oil cysts,and increased tissue integrity in several gel formulations (FIGS. 4B-4D)compared with control (FIG. 4A).

Example 17 Enhanced Angiogenesis in Fat Grafts Blended with HyaluronicAcid-Collagen(I) Hydrogels

Samples of hyaluronic acid-collagen(I) were implanted in a nude mousemodel for evaluation of angiogenic potential. Gels were implanted as abolus subcutaneously on the dorsum of female 6-week-old nude mice underanesthesia with two injections per mouse. Lipo was mixed with hydrogelsat a ratio of 2 parts lipo to 1 part gel (total 1 mL). Animals receivinglipo alone were used as controls (0.66 mL total fat injected). Six weekspost implantation, fat grafts were explanted and a subset used to assessangiogenesis levels by real-time PCR. Fat grafts used for real-time PCRwere placed in RNALater (Ambion) to protect RNA from degradationimmediately after being explanted. The RNALater treated tissue sampleswere lyophilized and pulverized before being used for RNA isolation.Total RNA was isolated (Aurum Total RNA Fatty and Fibrous kit, Bio-Rad)and its quality and quantity were assessed by Nanodrop and 1% agarosegel. The SuperScript VILO cDNA Synthesis kit (Invitrogen) was used forcDNA synthesis. The overall levels of angiogenesis were evaluated byCD31, an endothelial cell surface marker, gene expression levels byTaqman gene expression assay (Invitrogen) with StepOne Plus equipmentand Gene expression Analysis Software (Invitrogen). Human CD31expression in grafts with HA-Col(I) gels showed significantly higherlevels than lipoaspirate alone, more than 3 fold elevation (expressionfold change, Table 14), consistent with the previous finding thathyaluronic acid-collagen(I) hydrogels support endothelial cell survivaland proliferation in vitro.

TABLE 14 Human CD31 (PECAM) gene expression in fat grafts with andwithout HA- collagen(I) Sample (human CD31 Mean Expression Fold Changegene) N Relative to Lipo alone Lipo alone 5 1.00 ± 0.71 Lipo + HA-CN1 53.16 ± 0.55

Example 18 Enhanced Adipogenic Gene Expression in Fat Grafts Blendedwith Hyaluronic Acid-Collagen(I) Hydrogels

Samples of hyaluronic acid-collagen(I) were implanted in a nude mousemodel for evaluation of adipogenic potential. Gels were implanted as abolus subcutaneously on the dorsum of female 6-week-old nude mice underanesthesia with two injections per mouse. Lipo was mixed with hydrogelsat a ratio of 2 parts lipo to 1 part gel (total 1 mL). Animals receivinglipo alone were used as controls (0.66 mL total fat injected). Six weekspost implantation, fat grafts were explanted and a subset used to assessadipogenesis levels by real-time PCR. Fat grafts used for real-time PCRwere placed in RNALater (Ambion) to protect RNA from degradationimmediately after being explanted. The RNALater treated tissue sampleswere lyophilized and pulverized before being used for RNA isolation.Total RNA was isolated (Aurum Total RNA Fatty and Fibrous kit, Bio-Rad)and its quality and quantity were assessed by Nanodrop and 1% agarosegel. The SuperScript VILO cDNA Synthesis kit (Invitrogen) was used forcDNA synthesis. The overall adipogenesis levels of the fat grafts wereevaluated using adipocyte specific gene, leptin, expression at the RNAlevel. Grafts with HA-Col(I) gels showed a 2.5 fold increase in leptingene expression over the lipo alone samples (Table 15, p<0.05),suggesting an enhancement in adipogenic gene expression.

TABLE 15 Leptin gene expression in fat grafts with and withoutHA-collagen(I) Sample (human leptin Mean Expression Fold Change gene) NRelative to Lipo alone Lipo alone 4 1.00 ± 0.76 Lipo + HA-CN1 5 2.53 ±1.00

Example 19 Reduced Inflammation in Fat Grafts Blended with HyaluronicAcid-Collagen(I) Hydrogels

Samples of hyaluronic acid-collagen(I) were implanted in a nude mousemodel to evaluate the level of inflammation associated with these gels.Gels were implanted as a bolus subcutaneously on the dorsum of female6-week-old nude mice under anesthesia with two injections per mouse.Lipo was mixed with hydrogels at a ratio of 2 parts lipo to 1 part gel(total 1 mL). Animals receiving lipo alone were used as controls (0.66mL total fat injected). Six weeks post implantation, fat grafts wereexplanted and a subset used to assess general inflammation levels byreal-time PCR. Fat grafts used for real-time PCR were placed in RNALater(Ambion) to protect RNA from degradation immediately after beingexplanted. The RNALater treated tissue samples were lyophilized andpulverized before being used for RNA isolation. Total RNA was isolated(Aurum Total RNA Fatty and Fibrous kit, Bio-Rad) and its quality andquantity were assessed by Nanodrop and 1% agarose gel. The SuperScriptVILO cDNA Synthesis kit (Invitrogen) was used for cDNA synthesis.

The inflammatory responses of fat and gel grafts were determined by theexpression level of three genes, CD68 (a general macrophage marker),CD11c (a marker for macrophages involved in M1 pro-inflammatorypathway), and CD163 (a marker for macrophages involved in M2anti-inflammatory pathway). The CD68 positive macrophage responserepresents total macrophages and overall macrophage related inflammatoryresponses. Tissue macrophages, however, can exist in differentactivation states: either pro-inflammatory classically activated byinterferon-γ or lipopolysaccharide, known as M1, or anti-inflammatoryalternatively activated by IL-13 or IL-4, known as M2. Depending onmicroenvironmental stimuli, M1- and M2-activated macrophages fulfilldifferent functions through the production of pro- or anti-inflammatoryfactors. M2 macrophage activation is known to be involved in repair andremodeling of tissues, and as such, represents a productive response tothe injection or implantation of a biomaterial.

The results of real-time PCR indicated that fat grafts containing HA-CN1exhibited reduced levels of CD68 (<2 fold) compared to lipo alonegrafts. While also not significant, the reduced expression of CD11c andhigher CD163 expression levels (Table 16), suggest that HA-CN1 mighthave some effects in reducing pro-inflammatory responses and enhancinganti-inflammatory responses. Importantly, CD68 expression in fat graftscontaining HA-CN1 were not elevated, compared to lipo alone controls,suggesting that the hydrogel itself was not augmenting any inflammatoryresponse associated with the grafting of tissue.

TABLE 16 CD68, CD11c, and CD163 gene expression in fat grafts with andwithout HA-collagen(I) Mean Expression Fold Change Relative to mousemouse mouse Lipo alone N CD68 CD11c CD163 Lipo alone 5   1 ± 0.23   1 ±0.31   1 ± 0.25 Lipo + HA-CN1 5 0.69 ± 0.23 0.62 ± 0.20 1.29 ± 0.34

Example 20 Adipose Tissue Transplant for Breast Defect Correction

This example illustrates the use of compositions and methods disclosedherein for a breast defect correction.

A 32-year-old woman presented with complaints that the medial portionsof her breast implants were visible, which accentuated the “bony”appearance of her sternum. In addition, she felt her breasts were toofar apart. Pre-operative evaluation of the person includes routinehistory and physical examination in addition to thorough informedconsent disclosing all relevant risks and benefits of the procedure. Thephysician evaluating the individual determines that she is a candidatefor a soft tissue replacement method using the compositions and methodsdisclosed herein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the lateral and medial thigh regions.The harvested area is injected subcutaneously with a standard tumescentfluid solution containing a saline solution, 0.5% lidocaine, and about0.001% epinephrine. Using an 11-blade scalpel, a small puncture wound ismade in order to transverse the dermis. The blade is turned 360 degreesto complete the wound. A two-holed blunt harvesting cannula (3 mm innerdiameter) connected to a vacuum pump at low negative pressure (0.5 atm)is then inserted into the subcutaneous adipose tissue plane. The cannulais then moved throughout the plane to disrupt the connective tissuearchitecture. The volume of aspirate obtained is about 300 mL. Theharvest adipose tissue is processed by centrifugation at 3,000 g for 3minutes to separate health adipocytes and regenerative cells from blood,infiltration fluid and cell debris.

A hyaluronic acid-collagen(I) gel, such as described in Example 1, ismixed with the processed adipose tissue. The amount of compound added isan amount sufficient to promote formation of a blood supply sufficientto support the transplanted tissue. This composition is then transferredto 3 mL syringes. One-holed blunt infiltration cannulas (3 mm innerdiameter) are used to place the adipose tissue subcutaneously over thelateral sternum and medial breast bilaterally, 70 mL on the right and 50mL on the left. The adipose tissue is administered in a tear likefashion to increase the surface area to volume ratio.

Alternatively, the adipose tissue is first administered into theindividual, and a hyaluronic acid-collagen(I) gel, such as described inExample 1, is subsequently administered into the same, or in thevicinity of, the region where the adipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately six months after the procedure,the soft tissue replacement appears to be stable and the breast volumehas not decreased to any noticeable degree.

Example 21 Adipose Tissue Transplant for Breast Augmentation

This example illustrates the use of compositions and methods disclosedherein for a breast augmentation.

A 28-year-old woman presented micromastia or breast hypoplasia.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for a softtissue replacement method using the compositions and methods disclosedherein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the lateral and medial thigh regions.Using a 10-blade scalpel, a small puncture wound is made in order totransverse the dermis. The blade is turned 360 degrees to complete thewound. A two-holed blunt harvesting cannula (3 mm inner diameter)connected to a syringe is then inserted into the subcutaneous adiposetissue plane. The cannula is then moved throughout the plane to disruptthe connective tissue architecture. The volume of aspirate obtained isabout 600 mL. The harvest adipose tissue is processed by centrifugationat 2,700 g for 5 minutes to separate healthy adipocytes and regenerativecells from blood, infiltration fluid and cell debris. The centrifugedadipose tissue is then washed once is a Ringer's saline solution withlactone.

A hyaluronic acid-collagen(I) gel, such as described in Example 1, ismixed with the processed adipose tissue. The amount of compound added isan amount sufficient to promote formation of a blood supply sufficientto support the transplanted tissue. This composition is then transferredto 10 mL syringes. One-holed blunt infiltration cannulas (3 mm innerdiameter) are used to place the adipose tissue subcutaneously usingaxillary, periareolar, and inframammary routes bilaterally, 190 mL onthe right and 245 mL on the left. The adipose tissue is administered ina tear like fashion to increase the surface area to volume ratio.

Alternatively, the adipose tissue is first administered into theindividual, and a hyaluronic acid-collagen(I) gel, such as described inExample 1, is subsequently administered into the same, or in thevicinity of, the region where the adipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure.

Example 22 Adipose Tissue Transplant for Breast Disorder

This example illustrates the use of compositions and methods disclosedherein for a breast disorder.

A 49-year-old woman presented with bilaterial tuberous breast deformity.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for a softtissue replacement method using the compositions and methods disclosedherein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the abdomen, buttock, lateral andmedial thigh, and trochanter regions. Using a 12-blade scalpel, a smallpuncture wound is made in order to transverse the dermis. The blade isturned 360 degrees to complete the wound. A two-holed blunt harvestingcannula (3 mm inner diameter) connected to a syringe is then insertedinto the subcutaneous adipose tissue plane. The cannula is then movedthroughout the plane to disrupt the connective tissue architecture. Thevolume of aspirate obtained is about 1,400 mL.

The harvested adipose tissue is divided into two, approximately equalportions. One portion is processed by gravity sedimentation to separatehealthy adipocytes and regenerative cells from blood, infiltration fluidand cell debris. The other portion is used to isolate regenerativecells. This portion is digested with 0.075% collagenase in bufferedsaline for 30 minutes on a shaker at 37° C. Regenerative cells are thenseparated from mature adipocytes and connective tissue by centrifugingat 800 g for 10 minutes. The pellet containing the regenerative cells isthen washed three times with buffered saline. The washed regenerativecells are then added back to the sediment purified adipose tissue.

A hyaluronic acid-collagen(III) gel made in accordance with methodsdescribed herein, is mixed with the processed adipose tissue. The amountof compound added is an amount sufficient to promote formation of ablood supply sufficient to support the transplanted tissue. Thiscomposition is then transferred to 10 mL syringes. One-holed bluntinfiltration cannulas (3 mm inner diameter) are used to place theadipose tissue subcutaneously in multiple planes axillary, periareolar,and inframammary routes bilaterally, 380 mL on the right and 370 mL onthe left. The adipose tissue is administered in a tear like fashion toincrease the surface area to volume ratio.

The individual is monitored for approximately 21 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately twelve months after theprocedure, the woman indicates that her quality of life has improved.

In another instance, the individual is monitored for approximately 7days. The physician evaluates the engrafted tissue and determines thatthe engraftment was successful. Both the woman and her physician aresatisfied with the results of the procedure because she looked younger.Approximately one month after the procedure, the woman indicates thather quality of life has improved.

Example 23 Adipose Tissue Transplant to Treat Stress UrinaryIncontinence

This example illustrates the use of compositions and methods disclosedherein for treating stress urinary incontinence.

A 55 year old man presents with urinary incontinence. Pre-operativeevaluation of the patient includes routine history and physicalexamination in addition to thorough informed consent disclosing allrelevant risks and benefits of the procedure. The physician evaluatingthe individual determines that he is a candidate for a soft tissuereplacement method using the compositions and methods disclosed herein.

To begin the procedure, adipose tissue is harvested from the man. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the abdomen, and lateral and medialthigh regions. Using a 12-blade scalpel, a small puncture wound is madein order to transverse the dermis. The blade is turned 360 degrees tocomplete the wound. A two-holed blunt harvesting cannula (3 mm innerdiameter) connected to a syringe is then inserted into the subcutaneousadipose tissue plane. The cannula is then moved throughout the plane todisrupt the connective tissue architecture. The volume of aspirateobtained is about 900 mL.

A hyaluronic acid-collagen(I) gel, such as described in Example 1, ismixed with the processed adipose tissue. The amount of compound added isan amount sufficient to promote formation of a blood supply sufficientto support the transplanted tissue. This composition is then transferredto 20 mL syringes. One-holed blunt infiltration cannulas (14-gauge) areused to place about 800 mL of adipose tissue transdermally into thebladder neck and proximal urethra regions.

Alternatively, the adipose tissue is first administered into theindividual, and A hyaluronic acid-collagen(I) gel, such as described inExample 1, is subsequently administered into the same, or in thevicinity of, the region where the adipose tissue was implanted.

The individual is monitored after the procedure. Approximately threedays after the transplant, he experiences a decreased frequency ofincontinence. Approximately one month after the procedure, theindividual indicates that his quality of life has improved. Thephysician evaluates the engrafted tissue and determines that thelong-term engraftment was successful.

Example 24 Adipose Tissue Transplant for Breast Defect Correction

This example illustrates the use of compositions and methods disclosedherein for a breast defect correction.

A 56-year-old woman presents with a surgically removed breast due tocancer. Pre-operative evaluation of the person includes routine historyand physical examination in addition to thorough informed consentdisclosing all relevant risks and benefits of the procedure. Thephysician evaluating the individual determines that she is a candidatefor a soft tissue replacement method using the compositions and methodsdisclosed herein.

To begin the procedure, a breast mound is formed using a TRAM-flapprocedure. In this procedure, a portion of abdomen tissue, includingskin, adipose tissue, minor muscles and connective tissues, is takenfrom the patient's abdomen and transplanted onto the breast site. Thistissue is then used to create a breast mound.

A hyaluronic acid-collagen(III) gel, such as described herein, is thenadministered into the breast mound region. The amount of compoundadministered is an amount sufficient to promote formation of a bloodsupply sufficient to support the transplanted tissue.

The individual is monitored for approximately 21 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately one month after the procedure,the woman indicates that her quality of life has improved. Subsequentsurgery is performed to create a nipple and areola.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained. At the veryleast, and not as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

The terms “a,” “an,” “the,” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of any claim. Nolanguage in the specification should be construed as indicating anynon-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments disclosed herein arenot to be construed as limitations. Each group member may be referred toand claimed individually or in any combination with other members of thegroup or other elements found herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Certain embodiments are described herein, including the best mode knownto the inventors for carrying out the invention. Of course, variationson these described embodiments will become apparent to those of ordinaryskill in the art upon reading the foregoing description. The inventorexpects skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise thanspecifically described herein. Accordingly, the claims include allmodifications and equivalents of the subject matter recited in theclaims as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof iscontemplated unless otherwise indicated herein or otherwise clearlycontradicted by context.

In closing, it is to be understood that the embodiments disclosed hereinare illustrative of the principles of the claims. Other modificationsthat may be employed are within the scope of the claims. Thus, by way ofexample, but not of limitation, alternative embodiments may be utilizedin accordance with the teachings herein. Accordingly, the claims are notlimited to embodiments precisely as shown and described.

What is claimed is:
 1. A method of improving fat graft volume retention, the method comprising: providing a hydrogel component, the hydrogel component comprising: water; a crosslinked macromolecular matrix comprising hyaluronic acid crosslinked to collagen via a plurality of crosslink units, wherein at least a portion of the crosslink units comprise an amide bond, an ester bond or both; and the crosslinked macromolecular matrix has a weight ratio of the hyaluronic acid to the collagen of 1:1 to 7:1; wherein said hydrogel component has a hyaluronic acid concentration of 6 mg/mL to 21 mg/mL and a collagen concentration of 3 mg/mL to 12 mg/mL; and blending the hydrogel component with a lipoaspirate to form a lipoaspirate/hydrogel graft, wherein the lipoaspirate/hydrogel graft has a lipoaspirate:hydrogel component volume ratio of 1:1 to 5:1; and administering the lipoaspirate/hydrogel graft into a soft tissue in an area on a body of an individual, wherein the lipoaspirate/hydrogel graft promotes fat graft survival in the soft tissue area on the body.
 2. The method of claim 1, wherein the soft tissue comprises a volume, and the administering of the lipoaspirate/hydrogel graft increases the soft tissue volume of the area on the body.
 3. The method of claim 2, wherein the administering of the lipoaspirate/hydrogel graft increases fat volume in the area on the body.
 4. The method of claim 3, wherein retention of the fat volume is increased as compared to administering the lipoaspirate alone.
 5. The method of claim 1, wherein the method reduces variability in retained fat graft volume retention obtained from the administered lipoaspirate/hydrogel graft in the individual as compared to injection of lipoaspirate alone.
 6. The method of claim 1, wherein the method reduces variability in retained fat graft volume retention obtained from the administered lipoaspirate/hydrogel graft between patients and between different lipoaspirate samples.
 7. The method of claim 1, wherein the lipoaspirate/hydrogel graft is injected intradermally or subcutaneously.
 8. The method of claim 1, wherein the hyaluronic acid comprises a molecular weight of about 0.25 MDa, about 0.5 MDa, about 0.75 MDa about 1 MDa, about 1.25 MDa, about 1.5 MDa, about 1.75 MDa, about 2 MDa, about 2.25 MDa, about 2.5 MDa, about 2.75 MDa, about 3.0 MDa, about 3.25 MDa, about 3.5 MDa, about 3.75 MDa, about 4.0 MDa, about 4.25 MDa, about 4.5 MDa, about 4.75 MDa or about 5.0 MDa.
 9. The method of claim 1, wherein the hydrogel component comprises between 3 mg/ml to 24 mg/ml hyaluronic acid.
 10. The method of claim 9, wherein the hydrogel component comprises between 12 mg/ml to 16 mg/ml hyaluronic acid.
 11. The method of claim 1, wherein the hydrogel component comprises between 1 mg/ml collagen to 40 mg/ml collagen.
 12. The method of claim 11, wherein the hydrogel component comprises between 6 mg/ml collagen to 12 mg/ml collagen.
 13. The method of claim 1, wherein the lipoaspirate is from the individual.
 14. The method of claim 1, wherein the lipoaspirate/hydrogel graft has a lipoaspirate:hydrogel component volume ratio of 1:1, 2:1, 3:1, 4:1 or 5:1.
 15. The method of claim 14, wherein the lipoaspirate/hydrogel graft has a lipoaspirate:hydrogel component volume ratio of 2:1.
 16. The method of claim 14, wherein the lipoaspirate/hydrogel graft has a lipoaspirate:hydrogel component volume ratio of 4:1.
 17. The method of claim 14, wherein the lipoaspirate/hydrogel graft has a lipoaspirate:hydrogel component volume ratio of 5:1.
 18. The method of claim 1, wherein the hydrogel component has a storage modulus value of between a range of 850 Pa and 5,000 Pa.
 19. The method of claim 1, wherein the hyaluronic acid is crosslinked to the collagen.
 20. The method of claim 1, wherein the collagen is porcine collagen type I or human collagen type I.
 21. The method of claim 1, wherein the method improves tissue morphology.
 22. The method of claim 1, wherein the method allows diffusion of adipose tissue-specific growth factors or pro-antiogenic growth factors.
 23. The method of claim 1, wherein the method promotes cell and/or tissue growth.
 24. The method of claim 1, wherein the method stimulates angiogenesis, neovascularization, adipogenesis, and/or collagenesis.
 25. A method of enhancing or promoting cell proliferation or survival of one or more cells, the method comprising: administering a lipoaspirate/hydrogel graft to a body part of an individual, the lipoaspirate/hydrogel graft comprising: a hydrogel component, wherein the hydrogel component comprises: water; a crosslinked macromolecular matrix comprising hyaluronic acid crosslinked to collagen via a plurality of crosslink units, wherein at least a portion of the crosslink units comprise an amide bond, an ester bond or both; and the crosslinked macromolecular matrix has a weight ratio of the hyaluronic acid to the collagen of 1:1 to 7:1; wherein said hydrogel component has a hyaluronic acid concentration of 6 mg/mL to 21 mg/mL and a collagen concentration of 3 mg/mL to 12 mg/mL; and a lipoaspirate, wherein the lipoaspirate is from the individual, wherein administering enhances or promotes the cell proliferation or survival of one or more cells.
 26. The method of claim 25, wherein the cells comprises adipocytes, adipose-derived stem cells, stromal vascular fraction cells, or a combination thereof.
 27. A method for promoting cell survival comprising: administering a hydrogel graft to a body part of an individual, the hydrogel graft comprising: a hydrogel component, wherein the hydrogel component comprises: water; a crosslinked macromolecular matrix comprising hyaluronic acid crosslinked to collagen via a plurality of crosslink units, wherein at least a portion of the crosslink units comprise an amide bond, an ester bond or both; and the crosslinked macromolecular matrix has a weight ratio of the hyaluronic acid to the collagen of 1:1 to 7:1; and wherein said hydrogel component has a hyaluronic acid concentration of 6 mg/mL to 21 mg/mL and a collagen concentration of 3 mg/mL to 12 mg/mL.
 28. The method of claim 25, wherein the hydrogel graft enhances cell survival and proliferation of one or more cells.
 29. The method of claim 25, wherein the hyaluronic acid and collagen are crosslinked.
 30. The method of claim 27, wherein the hydrogel graft improves diffusion of adipose tissue-specific growth factors or pro-angiogenic growth factors into the body part that has received the hydrogel graft.
 31. A method of repairing a post-surgical defect, the method comprising providing a hydrogel component, the hydrogel component comprising: water; a crosslinked macromolecular matrix comprising hyaluronic acid crosslinked to collagen via a plurality of crosslink units, wherein at least a portion of the crosslink units comprise an amide bond, an ester bond or both; and the crosslinked macromolecular matrix has a weight ratio of the hyaluronic acid to the collagen of 1:1 to 7:1; wherein said hydrogel component has a hyaluronic acid concentration of 6 mg/mL to 21 mg/mL and a collagen concentration of 3 mg/mL to 12 mg/mL; and blending the hydrogel component with a lipoaspirate to form a lipoaspirate/hydrogel graft, wherein the lipoaspirate/hydrogel graft has a lipoaspirate:hydrogel component volume ratio of 1:1 to 5:1; and administering the lipoaspirate/hydrogel graft into an area on a body of an individual, wherein the lipoaspirate/hydrogel graft repairs or corrects a post-surgical defect in the area on the body.
 32. The method of claim 31, wherein the method corrects a post-surgical defect in breast tissue. 